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Sample records for igg antibodies assay

  1. Immunoradiometric assay for cytomegalovirus-specific IgG antibodies

    International Nuclear Information System (INIS)

    Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J.

    1990-01-01

    An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab

  2. A three-layer immunoradiometric assay for determination of IgG subclass antibodies in Human Sera (''IgG subclass RAST'')

    International Nuclear Information System (INIS)

    Djurup, R.; Soendergaard, I.; Weeke, B.; University of Copenhagen, Denmark); Magnusson, C.G.M.

    1984-01-01

    We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses, and the third layer is 125 I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgGI, anti-IgG3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-IgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Non-specific binding obtained with pooled normal human serum was below 0.33%. Inter-assay coefficient of variation was between 18 and 27%. The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy. (author)

  3. Immunoradiometric assay for cytomegalovirus-specific IgG antibodies; Assay development and evaluation in blood transfusion practice

    Energy Technology Data Exchange (ETDEWEB)

    Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J. (Medical School, Manchester (United Kingdom). Department of Medical microbiology, Virology Unit); Morell, G. (Regional Blood Transfusion Centre, manchester (United Kingdom))

    1990-03-01

    An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 (human cytomegalovirus (CMV)) has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab.

  4. A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

    International Nuclear Information System (INIS)

    Haas, G.G. Jr.; D'Cruz, O.J.

    1989-01-01

    Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM

  5. Performance Evaluation of the VIDAS® Measles IgG Assay and Its Diagnostic Value for Measuring IgG Antibody Avidity in Measles Virus Infection

    Science.gov (United States)

    Dina, Julia; Creveuil, Christian; Gouarin, Stephanie; Viron, Florent; Hebert, Amelie; Freymuth, Francois; Vabret, Astrid

    2016-01-01

    The objective of this study is primarily to compare the performance of the VIDAS® Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost® Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA® (Microimmune). The sensitivity and the agreement of the VIDAS® Measles IgG assay compared to the Enzygnost® Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA® assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS® Measles IgG assay to measure anti-measles virus IgG antibody avidity with the help of the VIDAS® CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI) 0.6. The VIDAS® Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects. PMID:27556477

  6. Immunochemical characteristics of IgG4 antibodies

    NARCIS (Netherlands)

    van der Zee, J. S.; Aalberse, R. C.

    1988-01-01

    Although a small part of the IgG4 subclass probably can bind to basophils (and mast cells), IgG4 antibodies usually do not behave as anaphylactic antibodies. Therefore, detection of IgG4 antibodies in serum is not a suitable in vitro assay for IgG-S-TS activity. Furthermore, differences between IgG4

  7. Anti-dog IgG secondary antibody successfully detects IgG in a variety of aquatic mammals

    Science.gov (United States)

    Roehl, Katherine; Jankowski, Mark D.; Hofmeister, Erik K.

    2016-01-01

    Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter (Enhydra lutris), polar bear (Ursus maritimus), grey seal (Halichoerus grypus), harbor seal (Phoca vitulina), northern elephant seal (Mirounga angustirostris), California sea lion (Zalophus californianus), Pacific walrus (Odobenus rosmarus) and one freshwater mammal: Asian small-clawed otter (Aonyx cinerea). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

  8. Timothy-specific IgG antibody levels vary with the pollen seasons.

    Science.gov (United States)

    Nordvall, S L; Larsson, P H; Johansson, S G

    1986-11-01

    Serum samples were collected from eight grass pollen hypersensitive children during a 4-year period. The sera were assayed for contents of timothy-specific IgE antibodies by RAST. Timothy-specific IgG and IgA antibodies were quantified by a refined ELISA in which covalent binding of the antigen to the polystyrene solid phase had been performed. IgG antibodies were also assayed by a Sepharose-protein-A technique with radiolabelled timothy allergens as the antigen. It was possible to register clearcut seasonal variations with postseasonally boosted antibody levels not only of timothy-specific IgE but also of IgG antibody. Both IgG1 and IgG4 antibodies specific for timothy showed seasonal variations of a similar degree. It was not possible to register seasonal variations of the same magnitude of timothy-specific IgA antibodies.

  9. Sensitive assays enable detection of serum IgG antibodies against Clostridium difficile toxin A and toxin B in healthy subjects and patients with Clostridium difficile infection.

    Science.gov (United States)

    Zhao, Xuemei; Bender, Florent; Shukla, Rajiv; Kang, John J; Caro-Aguilar, Ivette; Laterza, Omar F

    2016-04-01

    Pathogenic Clostridium difficile produces two proinflammatory exotoxins, toxin A and toxin B. Low level of serum antitoxin IgG antibodies is a risk factor for the development of primary and recurrent C. difficile infection (CDI). We developed and validated two sensitive, titer-based electrochemiluminescence assays for the detection of serum antibody levels against C. difficile toxins A and B. These assays demonstrated excellent precision. The sensitivity of the assays allowed the detection of antitoxin A and antitoxin B IgG antibodies in all tested serum samples during assay validation. The validated titer-based assays enable assessment of antitoxin A and antitoxin B IgG antibodies as potential biomarkers to identify patients with CDI at increased risk for CDI recurrence.

  10. EPSTEIN-BARR VIRUS INFECTIONS – AVIDITY TEST FOR IgG ANTIBODIES

    Directory of Open Access Journals (Sweden)

    Katja Strašek

    2001-06-01

    Full Text Available Background. We wish to introduce specific IgG avidity test as a supplementary assay in serological screening for Epstein-Barr virus infection if the status of patient cannot be resolved from a single serum sample with routine testing.Methods. Avidity of IgG antibodies was determined in sera of 57 patients with different stage of Epstein-Barr virus infection. Enzyme-immuno assay was used with a short incubation of 6-molar urea included in the procedure. Urea should remove low avidity antibodies. Avidity was expressed as the avidity index. Avidity testing with commercial kit was done as well.Results. Low avidity index was found for IgG antibodies of acute phase sera and high for those of past infection, recent infection and reactivation of endogenic virus.Conclusions. Avidity test for IgG antibodies might be supplementary assay to prove acute infection but also to resolve some other clinical states related to Epstein-Barr virus.

  11. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    International Nuclear Information System (INIS)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

    1982-01-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

  12. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

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    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  13. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    Science.gov (United States)

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

  14. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  15. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

    Science.gov (United States)

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

    2015-03-20

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    International Nuclear Information System (INIS)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-01-01

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

  17. A methodological approach for production and purification of polyclonal antibody against dog IgG.

    Science.gov (United States)

    Sadeghi, Somayeh; Aghebati-Maleki, Leili; Nozari, Samira; Majidi, Jafar

    2018-01-01

    Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens. In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

  18. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

    Science.gov (United States)

    DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

    2010-01-01

    Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

  19. Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis.

    Science.gov (United States)

    Baxter, C G; Denning, D W; Jones, A M; Todd, A; Moore, C B; Richardson, M D

    2013-04-01

    Detection of Aspergillus IgG antibodies is important in the diagnosis of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Immunoprecipitation techniques to detect these antibodies appear to lack sensitivity and accurate quantitation compared with enzyme immunoassays (EIA). This study assessed the performance of two commercial EIAs compared with counterimmunoelectrophoresis (CIE). This was a prospective cohort study of 175 adult patients with chronic or allergic pulmonary aspergillosis. Aspergillus IgG antibodies were detected using CIE, Phadia ImmunoCap Aspergillus IgG and Bio-Rad Platelia Aspergillus IgG. Inter-assay reproducibility was determined for each method and 25 patients had two serum samples analysed within a 6-month interval. When compared with CIE, both ImmunoCap and Platelia Aspergillus IgG had good sensitivity (97 and 93%, respectively) for detection of Aspergillus IgG antibodies. The level of agreement between the two EIAs for positive results was good, but the concentration of antibodies was not correlated between the tests or with CIE titre. ImmunoCap IgG inter-assay coefficient of variation was 5%, whereas Platelia IgG was 33%. Median ImmunoCap IgG values for CPA and allergic aspergillosis were 95 and 32 mg/L, respectively, whereas Platelia IgG values were >80 and 6 AU/mL. The direction of CIE titre change over 6 months was mirrored by ImmunoCap IgG levels in 92% of patients, and by Platelia IgG in 72% of patients. Both ImmunoCap and Platelia Aspergillus IgG EIAs are sensitive measures of Aspergillus IgG antibodies compared with CIE. However, ImmunoCap appears to have better reproducibility and may be more suitable for monitoring patient disease. © 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

  20. Radioimmunoassay of IgM, IgG, and IgA brucella antibodies

    International Nuclear Information System (INIS)

    Parrett, D.; Nielson, K.H.; White, R.G.; Payne, D.J.H.

    1977-01-01

    A radioimmunoassay (R.I.A.) has been devised to measure the serum antibody against Brucella abortus in each of the immunoglobulin classes IgM, IgG, and IgA. This test was applied to 46 sera from individuals with various clinical types of brucellosis, and the results were compared with the results of conventional direct and indirect agglutination and complement-fixation tests. The R.I.A. provided a highly sensitive primary-type assay which avoided the difficulties with blocking or non-agglutinating antibody, and thus has many advantages in the diagnosis of acute and chronic stages of brucella infection in man. The R.I.A. was successful in detection of antibody in many instances in which conventional serological tests were negative, and such antibody could (if IgM) be associated with acute or (if IgG or IgA) with chronic cases of brucellosis. One case in which B.abortus was isolated by blood culture but which failed to yield antibody by conventional tests, nevertheless showed substantial levels of IgM and IgG antibody by R.I.A. In other cases the R.I.A. test helped to eliminate the diagnosis of brucellosis by revealing absent or low antibody levels. (author)

  1. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

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    Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

    1982-12-01

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

  2. Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

    NARCIS (Netherlands)

    van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

    1986-01-01

    Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophagoïdes pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound

  3. Evaluation of cysticercus-specific IgG (total and subclasses and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    Full Text Available In the present study, an enzyme-linked immunosorbent assay (ELISA standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses and IgE antibodies in cerebrospinal fluid (CSF samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA=1.17 and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49 and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46 and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12 and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85 and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60 and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

  4. Production and assay of forskolin antibodies

    International Nuclear Information System (INIS)

    Ho, L.T.; Ho, R.J.

    1986-01-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3 H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3 H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC 50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

  5. Measurement of IgG antibodies to house dust mite and grass pollen by a solid-phase radioimmunoassay

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    Delespesse, G; Debisschop, M J; Flament, J [Hopital Saint Pierre, Louvain (Belgium). Lab. de Recherches de la Clinique Medicale

    1979-09-01

    A solid-phase radioimmunoassay was used to measure specific IgG antibodies to either Dermatophagoides pteronyssinus (DPT) or grass pollens. Radiolabelled protein A from Staphylococcus aureus (SpA) was used to determine the IgG antibodies attached to the microtubes. The binding of IgG from either normal or allergic sera to DPT-coated tubes was antigen specific and mediated by the Fab fragment of the immunoglobulin. IgG antibodies from non-allergic serum competed with IgE antibodies to DPT. IgE antibodies did not significantly interfere with the assay. Indeed heating a reaginic serum resulted in a striking reduction of the (/sup 125/I) anti- IgE binding to allergen-coated tubes without modifying the (/sup 125/I)-SpA binding. Furthermore, filtration of a reaginic serum through Sephacryl S-200 separated a peak of IgE antibodies. The solid phase method was more sensitive than a double-antibody technique employing the same DPT extract as labelled antigen. Non-allergic subjects had less IgG antibodies to DPT or grass pollens than allergic patients. In untreated patients, there was a good correlation between levels of IgG and IgE antibodies to grass pollens but not to DPT. Patients hyposensitized to house dust mite had on the average three times more specific IgG antibodies than untreated cases. (author).

  6. Measurement of IgG antibodies to house dust mite and grass pollen by a solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Delespesse, G.; Debisschop, M.J.; Flament, J.

    1979-01-01

    A solid-phase radioimmunoassay was used to measure specific IgG antibodies to either Dermatophagoides pteronyssinus (DPT) or grass pollens. Radiolabelled protein A from Staphylococcus aureus (SpA) was used to determine the IgG antibodies attached to the microtubes. The binding of IgG from either normal or allergic sera to DPT-coated tubes was antigen specific and mediated by the Fab fragment of the immunoglobulin. IgG antibodies from non-allergic serum competed with IgE antibodies to DPT. IgE antibodies did not significantly interfere with the assay. Indeed heating a reaginic serum resulted in a striking reduction of the ( 125 I) anti- IgE binding to allergen-coated tubes without modifying the ( 125 I)-SpA binding. Furthermore, filtration of a reaginic serum through Sephacryl S-200 separated a peak of IgE antibodies. The solid phase method was more sensitive than a double-antibody technique employing the same DPT extract as labelled antigen. Non-allergic subjects had less IgG antibodies to DPT or grass pollens than allergic patients. In untreated patients, there was a good correlation between levels of IgG and IgE antibodies to grass pollens but not to DPT. Patients hyposensitized to house dust mite had on the average three times more specific IgG antibodies than untreated cases. (author)

  7. Comparison of in-house biotin-avidin tetanus IgG enzyme-linked-immunosorbent assay (ELISA) with gold standard in vivo mouse neutralization test for the detection of low level antibodies.

    Science.gov (United States)

    Sonmez, Cemile; Coplu, Nilay; Gozalan, Aysegul; Akin, Lutfu; Esen, Berrin

    2017-06-01

    Detection of anti-tetanus antibody levels is necessary for both determination of the immune status of individuals and also for planning preventive measures. ELISA is the preferred test among in vitro tests however it can be affected by the cross reacting antibodies. A previously developed in-house ELISA test was found not reliable for the antibody levels ≤1.0IU/ml. A new method was developed to detect low antibody levels correctly. The aim of the present study was to compare the results of the newly developed in-house biotin-avidin tetanus IgG ELISA test with the in vivo mouse neutralization test, for the antibody levels ≤1.0IU/ml. A total of 54 serum samples with the antibody levels of three different levels, =0.01IU/ml, 0.01-0.1IU/ml, 0.1-1IU/ml, which were detected by in vivo mouse neutralization test were studied by the newly developed in-house biotin-avidin tetanus IgG ELISA test. Test was validated by using five different concentrations (0.01IU/ml, 0.06IU/ml, 0.2IU/ml, 0.5IU/ml, 1.0IU/ml). A statistically significant correlation (r 2 =0.9967 p=0,001) between in vivo mouse neutralization test and in-house biotin-avidin tetanus IgG ELISA test, was observed. For the tested concentrations intra-assay, inter-assay, accuracy, sensitivity, specificity and coefficients of variations were determined as ≤15%. In-house biotin-avidin tetanus IgG ELISA test can be an alternative method to in vivo mouse neutralization method for the detection of levels ≤1.0IU/ml. By using in-house biotin-avidin tetanus IgG ELISA test, individuals with non protective levels, will be reliably detected. Copyright © 2017. Published by Elsevier B.V.

  8. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    Science.gov (United States)

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  9. Detection of IgG antibodies against Bordetella pertussis with 125I-protein A

    International Nuclear Information System (INIS)

    Wirsing von Koenig, C.H.; Finger, H.

    1981-01-01

    A method for the detection of IgG antibodies against Bordetella pertussis is described, based on the principle of 'sandwich' radioimmunoassay. 125 I protein A is used as radioactive tracer. The influence of amounts of antigen, antibody, radioactive tracer, incubation time and temperature were tested and the optimal conditions for the assay are described. The procedure offers a simple, quick, and sensitive method for detecting antibodies against B. pertussis. Application and limitation of the test are discussed. (orig.)

  10. Toxoplasmosis serology: an efficient hemagglutination procedure to detect IgG and IgM antibodies

    Directory of Open Access Journals (Sweden)

    M.E. Camargo

    1989-08-01

    Full Text Available In search of an efficient but simple, low cost procedure for the serodiagnosis of Toxoplasmosis, especially suited for routine laboratories facing technical and budget limitations as in less developed countries, the diagnostic capability of Hematoxo® , an hemagglutination test for toxoplasmosis, was evaluated in relation to a battery of tests including IgG- and IgM-immunofluorescence tests, hemagglutination and an IgM-capture enzymatic assay. Detecting a little as 5 I.U. of IgG antitoxoplasma antibodies, Hematoxo® showed a straight agreement as to reactivity and non-reactivity for the 443 non-reactive and the 387 reactive serum samples, included in this study. In 23 cases presenting a serological pattern of acute toxoplasmosis and showing IgM antibodies, Hematoxo® could detect IgM antibodies in 18, indicated by negativation or a significant decrease in titers as a result of treating samples with 2-mercapto-ethanol. However, a neat increase in sensitivity for IgM specific antibodies could be achieved by previously removing IgG from the sample, as demonstrated in a series of acute toxoplasmosis sera. A simple procedure was developed for this purpose, by reconstituting a lyophilized suspension of Protein A - rich Staphylococcus with the lowest serum dilution to be tested. Of low cost and easy to perform, Hematoxo® affords not only a practical qualitative procedure for screening reactors and non-reactors, as in prenatal services, but also quantitative assays that permit to titrate antibodies as well as to identify IgM antibodies.

  11. Seroprevalence of dengue IgG antibodies in symptomatic and asymptomatic individuals three years after an outbreak in Zhejiang Province, China.

    Science.gov (United States)

    Luo, Shuying; Cui, Weihong; Li, Chan; Ling, Feng; Fu, Tao; Liu, Qiyong; Ren, Jiangping; Sun, Jimin

    2018-02-23

    Cross-reacting antibodies enhanced dengue infection in humans and antibody dependent enhancement (ADE) have been proposed as early mechanisms underlying DHF/DSS. However, the duration of dengue IgG antibodies in the body as well as factors associated with said duration remain unclear. Blood samples from 59 dengue symptomatic persons and 48 asymptomatic individuals were collected. Study participant demographic information (including age in 2009, gender, and place of residence) were also collected. Serum samples were tested for dengue specific IgG by Panbio dengue IgG indirect enzyme-linked immunosorbent assay (ELISA). Chi-square tests and logistic regression analysis of dengue IgG antibodies seroprevalence divided by gender, age groups, and symptomatic or asymptomatic infection were conducted using the Statistical Package for the Social Sciences. Overall, 70 (65.42%) blood samples were seropositive for dengue IgG antibodies with similar seroprevalences found when dividing by gender and different age groups. However, seroprevalence of dengue IgG antibodies in samples from dengue symptomatic persons was significantly higher than that in samples from asymptomatic individuals (96.61% vs 27.08%) according to multivariable logistic regression analysis, the odds ratio (OR) of the factor was 76.731. Dengue IgG antibodies were detectable in samples from most individuals three years after infection. Dengue symptomatic persons had a higher dengue IgG prevalence compared to asymptomatic individuals.

  12. Prevalence of Cytomegalovirus IgG Antibodies among Pregnant Women Visiting Antenatal Clinic, LAUTECH Teaching Hospital in Osogbo, Osun State, Nigeria.

    Science.gov (United States)

    Akende, Oluwatosin; Akanbi, Olusola Anuoluwapo; Oluremi, Adeolu Sunday; Okonko, Iheanyi Omezuruike; Opaleye, Oluyinka Oladele

    2016-01-01

    Cytomegalovirus (CMV) is one of the predominant viral infections that lead to congenital diseases and teratogenic risks during the perinatal stage. There is paucity of seroepidemiological data on anti-CMV IgG antibody in pregnant women in Osogbo, Osun State, Nigeria. This study was aimed at determining the seroprevalence of Cytomegalovirus IgG antibody among pregnant women visiting antenatal clinic, LAUTECH Teaching Hospital, Osogbo, Nigeria. One hundred and seventy-four sera from the pregnant women were screened by Enzyme linked Immunosorbent Assay (ELISA) for cytomegalovirus (CMV) IgG antibody. Data analysis was done using SPSS software. In this study, 105 of the 174 pregnant women were seropositive for CMV IgG antibodies giving an antibody prevalence of 60%. There was no association found between CMV IgG seropositivity and the subjects' demographic characteristics, however, the 60.0% prevalence of CMV-IgG antibody observed amongst pregnant women in this study demands for vaccines and regular testing for the presence of CMV and its related risk factors in antenatal clinic.

  13. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  14. Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

    Directory of Open Access Journals (Sweden)

    Yine Hu

    2015-01-01

    Full Text Available The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p0.05. Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  15. Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

    DEFF Research Database (Denmark)

    Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje

    2013-01-01

    alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a non-destructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on two human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge...

  16. Multiplex assay (Mikrogen recomBead) for detection of serum IgG and IgM antibodies to 13 recombinant antigens of Borrelia burgdorferi sensu lato in patients with neuroborreliosis

    DEFF Research Database (Denmark)

    Dessau, Ram Benny; Møller, Jens K.; Kolmos, Birte

    2015-01-01

    A multiplex-bead-based assay for the detection of serum antibodies to Borrelia burgdorferi sensu lato was evaluated. The assay contained 13 different antigens in both the IgG and the IgM assay; thus, a total of 26 measurement results were available from each sample. A total of 49 Danish patients......, the construction of a diagnostic score, evaluation of the scoring method using an independent dataset and an assessment of the analytical quality of the multiplex assay. The VlsE IgG had the highest diagnostic value with an AUC (area under the curve) of 96% on the receiver operating characteristic curve. The Osp......C IgM had AUCs just above 80%. All the other antigens had both low quantitative reactivity and lower contrast in the patients with LNB compared to controls. The diagnostic value of the assay may be improved by using a logistic model giving a sensitivity of 90 and 79% for the specificities at 92 and 98...

  17. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays.

    Science.gov (United States)

    Matee, M I; Lyamuya, E F; Simon, E; Mbena, E C; Kagoma, C; Samaranayake, L P; Scheutz, F

    1996-05-01

    The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.

  18. Anti-pituitary antibodies against corticotrophs in IgG4-related hypophysitis.

    Science.gov (United States)

    Iwata, Naoko; Iwama, Shintaro; Sugimura, Yoshihisa; Yasuda, Yoshinori; Nakashima, Kohtaro; Takeuchi, Seiji; Hagiwara, Daisuke; Ito, Yoshihiro; Suga, Hidetaka; Goto, Motomitsu; Banno, Ryoichi; Caturegli, Patrizio; Koike, Teruhiko; Oshida, Yoshiharu; Arima, Hiroshi

    2017-06-01

    IgG4-related disease is a systemic inflammatory disease characterized by infiltration of IgG4-positive plasma cells into multiple organs, including the pituitary gland. Autoimmunity is thought to be involved in the pathogenesis of IgG4-related disease. The diagnosis of IgG4-related hypophysitis (IgG4-RH) is difficult because its clinical features, such as pituitary swelling and hypopituitarism, are similar to those of other pituitary diseases, including lymphocytic hypophysitis and sellar/suprasellar tumors. The presence and significance of anti-pituitary antibodies (APA) in IgG4-RH is unclear. In this case-control study, we used single indirect immunofluorescence on human pituitary substrates to assess the prevalence of serum APA in 17 patients with IgG4-RH, 8 control patients with other pituitary diseases (lymphocytic infundibulo-neurohypophysitis, 3; craniopharyngioma, 2; germinoma, 3), and 9 healthy subjects. We further analyzed the endocrine cells targeted by the antibodies using double indirect immunofluorescence. APA were found in 5 of 17 patients with IgG4-RH (29%), and in none of the pituitary controls or healthy subjects. The endocrine cells targeted by the antibodies in the 5 IgG4-RH cases were exclusively corticotrophs. Antibodies were of the IgG1 subclass, rather than IgG4, in all 5 cases, suggesting that IgG4 is not directly involved in the pathogenesis. Finally, antibodies recognized pro-opiomelanocortin in 2 of the cases. Our study suggests that autoimmunity is involved in the pathogenesis of IgG4-RH and that corticotrophs are the main antigenic target, highlighting a possible new diagnostic marker for this condition.

  19. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

    NARCIS (Netherlands)

    Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules(1), Fab-arm exchange with therapeutic antibodies has not been

  20. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

    NARCIS (Netherlands)

    Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    2009-01-01

    Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated

  1. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    International Nuclear Information System (INIS)

    Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

    1980-01-01

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively

  2. Seroprevalence of diphtheria toxoid IgG antibodies in children, adolescents and adults in Poland.

    Science.gov (United States)

    Zasada, Aleksandra A; Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek

    2013-11-19

    Recommendations for diphtheria immunization are to apply an effective primary immunization in infancy and to maintain immunity throughout life. Immunity against diphtheria depends primarily on antibody to the diphtheria toxin. This study evaluated the seroprevalence of IgG diphtheria antitoxin in sera of healthy children, adolescents and adults in Poland. A total of 1387 serum samples collected between 2010 and 2012 from individuals with ages ranging from 1 month to 85 years were investigated. Antibody concentrations were measured with an enzyme-linked immunosorbent assay (Anti-Diphtheria Toxoid ELISA IgG, Euroimmun, Germany). The results showed that among 1387 individuals examined, 547 (39.4%) had anti-diphtheria toxoid IgG antibody levels below 0.1 IU/ml (36.9% ≤ 18 years and 40.5% >18 years old, respectively). The 212 (50.8%) children and 542 (55.9%) adults showed only basic protection (0.1-1.0 IU/ml) and need immediate booster. High levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were found more often in children and adolescent (12.2%) than in adults (3.6%) and this was statistically significant (P 60 years old. Characteristically, in individuals > 40 years old high levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were not seen. There were no statistically significant differences in results in relation to gender. The present study showed inadequate immunity levels to diphtheria amongst the Polish population, especially in adults > 40 years old and children ≤ 2 years old. To prevent reemergence of diphtheria an information campaign reminding people about recommendations concerning diphtheria booster vaccination in adults should be conducted. Moreover, the immunogenicity of the DTP vaccine used in Poland should be verified.

  3. Does the Maternal Serum IgG Level during Pregnancy in Primary Antibody Deficiency Influence the IgG Level in the Newborn?

    Directory of Open Access Journals (Sweden)

    Vasantha Nagendran

    2015-01-01

    Full Text Available Purpose. To find out if the serum IgG level in the newborn baby was affected by low maternal serum IgG during pregnancy in two newly diagnosed primary antibody deficient patients. Method. Infant cord blood IgG level was compared with maternal IgG level in 2 mothers with newly diagnosed primary antibody deficiency, who declined replacement IgG treatment during pregnancy. Results. Both mothers delivered healthy babies with normal IgG levels at birth. Conclusions. The normal IgG levels and sound health in these 2 babies in spite of low maternal IgG throughout pregnancy raise interesting discussion points about maternofoetal immunoglobulin transport mechanisms in primary antibody deficiency.

  4. Hybrid IgG4/IgG4 Fc antibodies form upon 'Fab-arm' exchange as demonstrated by SDS-PAGE or size-exclusion chromatography

    NARCIS (Netherlands)

    Rispens, Theo; den Bleker, Tamara H.; Aalberse, Rob C.

    2010-01-01

    Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab + Fc) with a molecular weight of ca. 100 kDa. These antibodies can

  5. IgG and IgG subclasses antibody responses to rK39 in Leishmania donovani infections

    International Nuclear Information System (INIS)

    Daifalla, N.S.; El Hassan, A.M.

    1998-01-01

    Leishmania donovani infection cause a wide spectrum of human diseases ranging from self-healing subclinical infections to severe visceral leishmaniasis, post kal-azar dermal leishmaiasis, and mucosal leishmaiasis. The infection associated with high levels of anti-leishmania antibodies which offer a potential parameter for the serological diagnosis of L. donovani infection replacing the invasive parasitological methods. rK39, a cloned antigen of L. chagasis was reported to have high levels of anti-leishmania antibodies in Sudanese and American visceral leishmaniasis patients. In an assessment of rK39-ELISA in detecting L. donovani infection we found that the antigen detected visceral leishmaniasis, post kala-azar dermal leishmaniasis, and mucosal leismaniasis with the sensitives of 96.6%, 95.91% and 90.91% respectively. The test has the specificity of 96.7%. Further investigation of 25 visceral leishmaniasis patients showed elevated anti-rK39 antibody responses of IgG subclasses with IgG1 and IgG3 significantly higher than IgG4. igG3 showed the highest sensitivity (84.00%) whereas IgG1 showed the highest sensitivity (100%). The dynamics of the serological reactivity to rK39 in l.donovani infections will be discussed in relation to exposure, infection, cure and relapse.(Author)

  6. Generation and characterization of antibodies against Asian elephant (Elephas maximus IgG, IgM, and IgA.

    Directory of Open Access Journals (Sweden)

    Alan F Humphreys

    Full Text Available Asian elephant (Elephas maximus immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV, which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV.

  7. IgG isotypic antibodies to crude Plasmodium falciparum blood-stage ...

    African Journals Online (AJOL)

    Methods: Levels of IgG (IgG1-IgG4) and IgM to crude P. falciparum blood stage antigen ... dosage influenced P. falciparum-specific isotypic antibody responses to blood stage .... exposed Swedish donors. ..... with adverse pregnancy outcomes.

  8. Inhibition of complement activation by IgG4 antibodies

    NARCIS (Netherlands)

    van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

    1986-01-01

    Prolonged exposure to antigens may result in high IgG4 antibody titres as was shown in a previous paper (Aalberse et al., 1983b). In novice bee keepers, a shift in the IgG1/IgG4 ratio of the response against phospholipase-A (PLA; a major component of bee venom) occurred. This resulted in an

  9. IgG and IgE antibodies to Chironomidae in asthmatic patients.

    Science.gov (United States)

    Yamashita, N; Ito, K; Nakagawa, T; Haida, M; Okudaira, H; Nakada, S; Miyamoto, T; Shibuya, T; Kamei, K; Sasa, M

    1987-01-01

    IgG antibodies to Chironomidae and its correlations to radioallergosorbent and skin reactions were examined with the aim of clarifying the relationship between asthma and Chironomidae. The level of specific IgG antibody in asthmatic patients (0.698 +/- 0.034, n = 104) was significantly greater than that in normal subjects (0.367 +/- 0.032, n = 52) (P less than 0.01). The specific IgG level was not correlated to skin reaction, nor to IgE RAST scores. Specific IgG1 and IgG4 levels in asthmatic patients were significantly greater than in control subjects (n = 14) (P less than 0.01). Images Fig. 5 PMID:3652516

  10. Antibody isotypes, including IgG subclasses, in Ecuadorian patients with pulmonary Paragonimiasis

    Directory of Open Access Journals (Sweden)

    Angel Guevara E.

    1995-08-01

    Full Text Available An ELISA test was developed to detect Paragonimus-specific antibodies, including IgG subclasses, using P. mexicanus crude water-soluble antigens. The test was standardized to detect antibodies in sera of Ecuadorian patients with pulmonary paragonimiasis and negative controls from the endemic area. The detected mean levels of IgG (0.753, SEM: 0.074 and IgM (0.303, SEM: 0.033 were significantly elevated (P<0.05. Within the IgG subclasses, IgG4 showed the highest detected mean level (0.365, SEM: 0.116 and the other three subclasses showed considerably lower mean levels (IgG1, 0.186 SEM: 0.06; IgG2, 0.046 SEM: 0.01; IgG3, 0.123 SEM: 0.047. The number of P. mexicanus eggs found in sputum of infected individuals showed a positive correlation with the level of antibodies detected for IgM, IgG and its subclasses (P<0.001. The relevance of these findings in Ecuadorian patients suffering from pulmonary paragonimiasis is discussed.

  11. Development and characterization of highly informative ELISA for the detection of IgG and IgA antibodies to Сhlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2018-04-01

    Full Text Available The goal of this work was developing of highly informative an enzyme-linked immunosorbent assay (ELISA for the detection of IgG and IgA antibodies against to Chlamydia trachomatis, as well as comparative characterization of developed assay using standardized control materials. The study was conducted using: monoclonal antibodies (McAbs to human IgA and IgG; recombinant Ch. trachomatis proteins – Pgp3, major outer membrane protein (MOMP; two panels of characterized sera and four reference ELISA kits. The study of immunochemical activity of peroxidase conjugates of McAbs was performed in comparison with conjugates of commercial analogues: anti-IgG McAb 2A11 and anti-IgA McAb AD3. About half of the conjugates from the received McAbs panel were more active compared to the reference antibody conjugates. It was quite justified to use the conjugates of antibodies that interact with different antigenic determinants. When IgG antibodies were detected to MOMP, it was justified 1.14-1.56 times more; when IgA antibodies were detected to MOMP, it was justified 1.16-1.37 times more. ELISA for detecting IgG/IgA antibodies to MOMP and Pgp3 of Ch. trachomatis were evaluated using appropriately described serum panels OCO-42-28-313-00 and OCO-42-28-314-00. Comparative studies of the developed ELISA for the detection of IgG and IgA antibodies to the MOMP and Pgp3 of Ch. trachomatis showed their prominent advantage over the commercial analogues, which more clearly demonstrates the difference in the ratio of average values of optical density of positive and negative samples of the described panel of sera: this indicator for commercial kits was 1.36-3.59 times less.

  12. A ''reverse'' solid-phase radio-immuno-assay for IgM-antibodies to hepatitis A virus

    International Nuclear Information System (INIS)

    Meurman, O.H.; Matter, L.; Krishna, R.V.; Krech, U.H.

    1981-01-01

    A ''reverse'' solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and 125 I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sera was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar ''reverse'' immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens. (author)

  13. Predictive value of Borrelia burgdorferi IgG antibody levels in patients referred to a tertiary Lyme centre.

    Science.gov (United States)

    Zwerink, M; Zomer, T P; van Kooten, B; Blaauw, G; van Bemmel, T; van Hees, B C; Vermeeren, Y M; Landman, G W

    2018-03-01

    A two-step testing strategy is recommended in serological testing for Lyme borreliosis; positive and indeterminate enzyme-linked immunosorbent assay (ELISA) results are confirmed with immunoblots. Several ELISAs quantify the concentration of antibodies tested, however, no recommendation exists for an upper cut-off value at which an IgG ELISA is sufficient and the immunoblot can be omitted. The study objective was to determine at which IgG antibody level an immunoblot does not have any additional predictive value compared to ELISA results. Data of adult patients who visited a tertiary Lyme centre between 2008 and 2014 were analysed. Both an ELISA (Enzygnost Lyme link VlsE IgG) and immunoblot (recomLine blot Borrelia) were performed. Clinical data were extracted from the patient's digital medical record. Positive predictive values (PPVs) for either previous or active infection with Borrelia burgdorferi s.l. were calculated for different cut-off ELISA IgG antibody levels where the immunoblot was regarded as reference test. In total, 1454 patients were included. According to the two-step test strategy, 486 (33%), 69 (5%) and 899 (62%) patients had positive, indeterminate and negative Borrelia IgG serology, respectively. At IgG levels of 500 IU/ml and higher, all immunoblots were positive, resulting in a 100% PPV (95% CI: 97.0-100). At IgG levels of 200 IU/ml and higher, the PPV was 99.3% (95% CI: 97.4-99.8). In conclusion, at IgG levels of 200 IU/ml and higher, an ELISA was sufficient to detect antibodies to Borrelia burgdorferi s.l. At those IgG levels, a confirmatory immunoblot may be omitted in patients referred to a tertiary Lyme centre. Before these results can be implemented in routine diagnosis of Lyme borreliosis, confirmation of the results is necessary in other patient populations and using other quantitative ELISAs and immunoblots. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. IgG4 subclass antibodies impair antitumor immunity in melanoma

    Science.gov (United States)

    Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

    2013-01-01

    Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

  15. A new and rapid method for immunoglobulin class and subclass determination of mouse monoclonal antibodies using a solid-phase immunoradiometric assay

    International Nuclear Information System (INIS)

    Storch, M.-J.; Lohmann-Matthes, M.-L.

    1984-01-01

    A solid-phase immunoradiometric assay is described for the detection of mouse immunoglobulin classes and subclasses in unpurified and unconcentrated supernatants of hybridomas. IgG fractions from rabbit antisera specific for mouse immunoglobulin classes and subclasses are used for coating the wells of flexible microtiter plates. Monoclonal antibody present in hybridoma supernatants is bound only to wells that contain the appropriate anti-subclass antibody. The binding of hybridoma antibodies to corresponding IgG subclasses or IgM is then detected by a labeled rabbit anti-mouse antibody binding to all mouse immunoglobulins (heavy and light chains). Thus, only 1 labeled antibody is needed for all assays. The advantages of the method described are the following: results are obtained within a few hours and antibody containing hybridoma supernatants may be used without a concentration step since minute amounts of antibody are detected by the immunoradiometric assay. Cultures producing several subclasses may be early recognized as oligo/polyclonal. (Auth.)

  16. High Maternal HIV-1 Viral Load During Pregnancy Is Associated With Reduced Placental Transfer of Measles IgG Antibody

    Science.gov (United States)

    Farquhar, Carey; Nduati, Ruth; Haigwood, Nancy; Sutton, William; Mbori-Ngacha, Dorothy; Richardson, Barbra; John-Stewart, Grace

    2012-01-01

    Background Studies among HIV-1–infected women have demonstrated reduced placental transfer of IgG antibodies against measles and other pathogens. As a result, infants born to women with HIV-1 infection may not acquire adequate passive immunity in utero and this could contribute to high infant morbidity and mortality in this vulnerable population. Methods To determine factors associated with decreased placental transfer of measles IgG, 55 HIV-1–infected pregnant women who were enrolled in a Nairobi perinatal HIV-1 transmission study were followed. Maternal CD4 count, HIV-1 viral load, and HIV-1–specific gp41 antibody concentrations were measured antenatally and at delivery. Measles IgG concentrations were assayed in maternal blood and infant cord blood obtained during delivery to calculate placental antibody transfer. Results Among 40 women (73%) with positive measles titers, 30 (75%) were found to have abnormally low levels of maternofetal IgG transfer (<95%). High maternal HIV-1 viral load at 32 weeks’ gestation and at delivery was associated with reductions in placental transfer (P < 0.0001 and P = 0.0056, respectively) and infant measles IgG concentrations in cord blood (P < 0.0001 and P = 0.0073, respectively). High maternal HIV-1–specific gp41 antibody titer was also highly correlated with both decreased placental transfer (P = 0.0080) and decreased infant IgG (P < 0.0001). Conclusions This is the first study to evaluate the relationship between maternal HIV-1 viremia, maternal HIV-1 antibody concentrations, and passive immunity among HIV-1–exposed infants. These data support the hypothesis that high HIV-1 viral load during the last trimester may impair maternofetal transfer of IgG and increases risk of measles and other serious infections among HIV-1–exposed infants. PMID:16280707

  17. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhenbao [Central South University, School of Pharmaceutical Sciences (China); Zhou, Bo, E-mail: zhoubo1771@163.com [The Affiliated Zhongda Hospital of Southeast University, Department of Gerontology (China); Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang, E-mail: lengxigyky@163.com [Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College (China)

    2013-09-15

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 {mu}m in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL{sup -1} to 10 ng mL{sup -1}. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  18. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Science.gov (United States)

    Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

    2013-09-01

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  19. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    International Nuclear Information System (INIS)

    Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

    2013-01-01

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL −1 to 10 ng mL −1 . This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples

  20. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti......-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells....

  1. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

    Science.gov (United States)

    Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

    2015-01-01

    Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Seroprevalence of parvovirus B19 IgG and IgM antibodies among pregnant women in Oyo State, Nigeria.

    Science.gov (United States)

    Abiodun, Iyanda; Opaleye, Oluyinka Oladele; Ojurongbe, Olusola; Fagbami, Ademola Hezekiah

    2013-12-15

    Human parvovirus B19 causes a wide range of complications in pregnant women including abortion, severe fetal anemia, non-immune hydrops fetalis, and even intrauterine fetal death. However, there is a dearth of information on the prevalence of the virus among pregnant women in southwestern Nigeria. Blood samples were collected from 231 pregnant women and screened for antibodies to human parvovirus B19 IgM and IgG using an enzyme immunosorbent assay kits. Of the 231 women, 31 were in their first trimester, 146 were in their second trimester, and 54 were in their third trimester. Forty-five (20%) were positive for parvovirus B19 IgG antibodies, 10 (4%) were positive for parvovirus B19 IgM antibodies, and 176 (76%) had no detectable parvovirus B19 antibodies. Twenty-eight (19%) of the 146 pregnant women in their second trimester were positive for parvovirus B19 IgG antibody while three (2%) of the 146 were positive for parvovirus B19 IgM antibody. It is evident that there is a high prevalence of human parvovirus B19 among pregnant women in south-western Nigeria. This suggests that there is an active transmission of the virus in the community; it is therefore necessary to conduct more studies on the virus in pregnant women in Nigeria to ascertain its effect on the fetus.

  3. Detection of phase I IgG antibodies to Coxiella burnetii with EIA as a screening test for blood donations.

    Science.gov (United States)

    van der Hoek, W; Wielders, C C H; Schimmer, B; Wegdam-Blans, M C A; Meekelenkamp, J; Zaaijer, H L; Schneeberger, P M

    2012-11-01

    The presence of a high phase I IgG antibody titre may indicate chronic infection and a risk for the transmission of Coxiella burnetii through blood transfusion. The outbreak of Q fever in the Netherlands allowed for the comparison of an enzyme immunoassay (EIA) with the reference immunofluorescence assay (IFA) in a large group of individuals one year after acute Q fever. EIA is 100 % sensitive in detecting high (≥1:1,024) phase I IgG antibody titres. The cost of screening with EIA and confirming all EIA-positive results with IFA is much lower than screening all donations with IFA. This should be taken into account in cost-effectiveness analyses of screening programmes.

  4. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

    Directory of Open Access Journals (Sweden)

    Xinsheng Tian

    2015-01-01

    Full Text Available IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.

  5. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A P; Rezapkin, G V; Dzagurova, T K; Tkachenko, E A [Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow

    1984-05-01

    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  6. Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein.

    Science.gov (United States)

    Kakalacheva, Kristina; Regenass, Stephan; Wiesmayr, Silke; Azzi, Tarik; Berger, Christoph; Dale, Russell C; Brilot, Fabienne; Münz, Christian; Rostasy, Kevin; Nadal, David; Lünemann, Jan D

    2016-02-12

    A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM.

  7. Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

    Science.gov (United States)

    Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

  8. Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

    Directory of Open Access Journals (Sweden)

    Kathryn L Armour

    Full Text Available We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

  9. Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

    Science.gov (United States)

    Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

    2017-09-01

    Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

  10. An enzyme-linked immuno-filtration assay used to compare infant and maternal antibody profiles in toxoplasmosis.

    Science.gov (United States)

    Pinon, J M; Thoannes, H; Gruson, N

    1985-02-28

    Enzyme-linked immuno-filtration assay is carried out on a micropore membrane. This doubly analytical technique permits simultaneous study of antibody specificity by immunoprecipitation and characterisation of antibody isotypes by immuno-filtration with enzyme-labelled antibodies. Recognition of the same T. gondii antigenic constituent by IgG, IgA, IgM or IgE antibodies produces couplets (IgG-IgM; IgG-IgA) or triplets (IgG-IgM-IgA; IgG-IgM-IgE) which identify the functional fractions of the toxoplasmosis antigen. In acquired toxoplasmosis, the persistence of IgM antibody long after infestation puts in question the implication of recent infestation normally linked to detection of this isotype. For sera of comparable titres, comparison of immunological profiles by the method described demonstrates disparities in the composition of the specific antibody content as expressed in international units. Use of the same method to detect IgM antibodies or distinguish between transmitted maternal IgG and IgG antibodies synthesised by the foetus or neonate makes a diagnosis of congenital toxoplasmosis possible in 85% of cases during the first few days of life. With the method described the diagnosis may be made on average 5 months earlier than with classical techniques. In the course of surveillance for latent congenital toxoplasmosis, the appearance of IgM or IgE antibodies raises the possibility of complications (hydrocephalus, chorioretinitis). After cessation of treatment, a rise in IgG antibodies indicating persistence of infection is detected earlier by the present than by classical methods.

  11. The Induction of IgM and IgG Antibodies against HLA or MICA after Lung Transplantation

    Directory of Open Access Journals (Sweden)

    Annelieke W. M. Paantjens

    2011-01-01

    Full Text Available The production of IgG HLA antibodies after lung transplantation (LTx is considered to be a major risk factor for the development of chronic rejection, represented by the bronchiolitis obliterans syndrome (BOS. It has recently been observed that elevated levels of IgM HLA antibodies also correlates with the development of chronic rejection in heart and kidney transplantation. This study investigates the relationship between IgM and IgG antibodies against HLA and MICA after lung transplantation. Serum was collected from 49 patients once prior to transplantation and monthly for up to 1 year after lung transplantation was analyzed by Luminex to detect IgM and IgG antibodies against HLA and MICA. The presence of either IgM or IgG HLA and/or MICA antibodies prior to or after transplantation was not related to survival, gender, primary disease, or the development of BOS. Additionally, the production of IgG alloantibodies was not preceded by an increase in levels of IgM, and IgM levels were not followed by an increase in IgG. Under current immune suppressive regimen, although the presence of IgM antibodies does not correlate with BOS after LTx, IgM high IgG low HLA class I antibody titers were observed more in patients with BOS compared to patients without BOS.

  12. Relation between IgG antibodies to foods and IgE antibodies to milk, egg, cat, dog and/or mite in a cross-sectional study

    NARCIS (Netherlands)

    Eysink, P. E.; de Jong, M. H.; Bindels, P. J.; Scharp-van der Linden, V. T.; de Groot, C. J.; Stapel, S. O.; Aalberse, R. C.

    1999-01-01

    Because IgG antibodies to foods can be detected before IgE antibodies to inhalants, increased levels of IgG antibodies to foods might be used as a predictor of IgE-mediated allergy in initially nonatopic children. To examine the cross-sectional relation between IgG to foods (i.e. mixture of wheat

  13. Chlamydial serum IgG, IgA and local IgA antibodies in patients with genital-tract infections measured by solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Terho, P.; Meurman, O.

    1981-01-01

    A solid-phase radioimmunoassay (RIA) for IgG and IgA class antibodies to Chlamydia trachomatis was developed with C. trachomatis serotype L2 as antigen. The assay was sensitive, reproducible and correlated well with an immunofluorescence test (r = 0.85). Serum IgG antibodies were detected in 79% of Chlamydia isolation-positive versus 43% of isolation-negative male patients with urethritis and serum IgA antibodies in 53% and 21%, respectively. Urethral IgA antibodies, measured from specimens taken for chlamydial isolation, could be detected in 94% and 38%, respectively. From 737 male urethral and 909 female cervical secretions screened for the presence of IgA antibodies, about half were isolation and IgA negative. Only 4% (6/151) of male and 5.4% (2/37) of female isolation-positive specimens were IgA negative. The determination of local IgA antibodies may be used as a screening test in chlamydial genital infections. (author)

  14. Comparison of Toxoplasma gondii IgG avidity Architect and Vidas assays with the estimated date of infection in pregnant women

    Directory of Open Access Journals (Sweden)

    Smets Aurélie

    2016-01-01

    Full Text Available A maternal Toxoplasma gondii infection during pregnancy is a risk for congenital infection through maternal-fetal transplacental transmission. Estimation of the date of infection is of the utmost importance for management and treatment recommendations. In this setting, IgG avidity has been shown to be useful as high avidity rules out an infection dating less than 4 months. The estimated date of infection can also be obtained by the ratio of T. gondii IgG titers measured by the Vidas (bioMérieux assay versus T. gondii IgG titers measured by the Architect (Abbott Laboratories test, together with T. gondii IgM and IgA antibody responses. In this study, using 117 serum samples from pregnant women, we compared the IgG avidity values obtained by Architect and Vidas with the presumed date of T. gondii infection established by the T. gondii IgG ratio of IgG Vidas and IgG Architect plus the IgM and IgA results. To date, IgG avidity Vidas seems to exhibit better performance than Architect. For both assays, gray zone results were most likely obtained from patients infected more than 4 months before sampling. These data should be taken into account for a possible reconsideration of the interpretation of avidity results in the gray zone.

  15. Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

    International Nuclear Information System (INIS)

    Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  16. Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

    Directory of Open Access Journals (Sweden)

    R. Sharma

    2010-02-01

    Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

  17. Human IgG subclass antibodies to the 19 kilodalton carboxy ...

    African Journals Online (AJOL)

    IgG2 or IgG4 antibodies were virtually nonexistent. The cross-reactivity between the 4 sequence variants (E-KNG, E-TSR, Q-KNG and. Q-TSR) of MSP119 was confirmed; however, a minority of sera preferentially recognised the KNG but not the TSR variants. All 33 P. falciparum isolates from different parts ofm Uganda

  18. Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG

    International Nuclear Information System (INIS)

    Hibma, M.; Griffin, J.F.T.

    1992-01-01

    Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species

  19. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange

    NARCIS (Netherlands)

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K.; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H.; Wiegman, Luus; Vink, Tom; Aarden, Lucien A.; de Baets, Marc H.; van de Winkel, Jan G. J.; Aalberse, Rob C.; Parren, Paul W. H. I.

    2007-01-01

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4

  20. Determination of Antibodies (IgG, IgM against Toxoplasma gondii in Patients with Cancer

    Directory of Open Access Journals (Sweden)

    M Pedram

    2007-08-01

    Full Text Available Background: The aim of this study was determination of antibodies (IgG, IgM against Toxoplasma in malignant patients in order to refer the patients on time to the physician for treatment.Methods: This study was carried out on 252 malignant patients and 252 healthy normal subjects (as control obtained from Shafa Hospital and Medical Diagnostic Laboratory (Iran-Zamin, in Ahwaz city. Patient's information was recorded in a questionnaire before sampling. Serum samples of patients were examined for IgG and IgM antibodies by ELISA technique using Trinity kits. Results: The results of this study revealed the presence of Toxoplasma antibodies in 114 (45.2% cases of patients who were positive for Toxoplasma IgG antibodies, and 26 (10.3% cases were confirmed to be positive for Toxoplasma IgM antibodies and also 17 (6.7% of cases had both IgG and IgM antibodies against Toxoplasma gondii. In control group 92 (36.5% cases and 15 (6% cases revealed seropositive for IgG and IgM antibodies, respectively. There were no significant differences between sex, close contact with cat, living region, chemotherapy, and seropositivity rate of toxoplasmosis in patients. Comparing the age groups, the highest seropositive rate showed in the age of 51 years or higher, and their rates had tendency to increase with age in both groups. No seropositivity significant relationship was found between patients and control group.Conclusion: According to the prevalence of positive cases in these patients, it is necessary to examine the patients for toxoplasmosis before, during and after chemotherapy.

  1. High seropositivity of IgG and IgM antibodies against ...

    African Journals Online (AJOL)

    Objective: This study reports on the high seropositivity of immunoglobulin (Ig) G and M antibodies against CMV and the risk factors for CMV ... sex, highly active antiretroviral therapy (HAART) were not statistically associated with CMV seropositivity in this study. ... are infected with HIV have detectable IgG antibodies to CMV ...

  2. Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

    Science.gov (United States)

    Alderete, J F

    1984-06-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.

  3. Merkel cell polyomavirus IgG antibody levels are associated with progression to AIDS among HIV-infected individuals.

    Science.gov (United States)

    Vahabpour, Rouhollah; Nasimi, Maryam; Naderi, Niloofar; Salehi-Vaziri, Mostafa; Mohajel, Nasir; Sadeghi, Farzin; Keyvani, Hossein; Monavari, Seyed Hamidreza

    2017-04-01

    The association of Merkel cell polyomavirus (MCP y V) with Merkel cell carcinoma (MCC) in immunocompromised individuals has been revealed in a number of surveys. The study of MCP y V specific antibody titers and viral loads in such patients has a great attraction for research groups interested in viral reactivation. In this cross-sectional study to evaluate MCP y V antibody titer, DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs), we examined 205 HIV-1 infected patients and 100 un-infected controls. The HIV-1 infected patients divided into two groups (HIV/AIDS and non-AIDS) according to their CD4 status. Total IgG antibody titer against MCP y V was analyzed by virus like particle (VLP)-based enzyme linked immunosorbent assay (ELISA). Presence of MCP y V-DNA in subject's PBMCs was examined by quantitative real-time PCR assay. Levels of anti-MCP y V IgG in HIV/AIDS patients were significantly higher than those in non-AIDS HIV-infected and control subjects (p value = <0.001). The prevalence rate of MCP y V-DNA in PBMCs of HIV/AIDS, non-AIDS HIV-infected and un-infected controls were 17%, 16%, and 14% respectively. The MCP y V viral load among the groups ranged between 0.15 to 2.9 copies/10 3 cells (median, 1.9 copies/10 3 cells), with no significant difference between the studied populations (p value = 0.3).

  4. Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector Borne Diseases: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Berlin L. Londono-Renteria

    2016-09-01

    Full Text Available Chronic exposure to antigens may favor the production of IgG4 antibodies over other antibody types. Recent studies have shown that up to a 30% of normal human IgG4 is bi-specific and is able to recognize two antigens of different nature. A requirement for this specificity is the presence of both eliciting antigens in the same time and at the same place where the immune response is induced. During transmission of most vector-borne diseases, the pathogen is delivered to the vertebrate host along with the arthropod saliva during blood feeding and previous studies have shown the existence of IgG4 antibodies against mosquito salivary allergens. However, there is very little ongoing research or information available regarding IgG4 bi-specificity with regards to infectious disease, particularly during immune responses to vector-borne diseases such as malaria, filariasis or dengue virus infection. Here, we provide background information and present our hypothesis that IgG4 may not only be a useful tool to measure exposure to infected mosquito bites, but that these bi-specific antibodies may also play an important role in modulation of the immune response against malaria and other vector-borne diseases in endemic settings.

  5. Seroprevalence of IgG Antibodies against Mumps in Bulgarian Children under 18 Years of Age

    Directory of Open Access Journals (Sweden)

    Milena Karcheva

    2013-03-01

    Full Text Available Objective: The objective of this study was to determine the prevalence of IgG antibodies against mumps in a representative stratified sample of children under 18 years of age in Pleven district, Bulgaria. Study Design: Cross sectional study. Materials and Methods: A cross-sectional, sero-epidemiologic survey of 132 children aged under 18 years was conducted in Pleven district. They were distributed into two age groups: from 0 to 11 years old (n=80 and from 12 to 18 years old (n=52. An enzyme immunoassay (EIA was performed for indirect evidence of specific IgG antibodies in the serum. Results: The results showed that 41% of the investigated children were negative in terms of mumps IgG antibodies, 11% had borderline values and the remaining, approximately 48%, were positive. In the group of children aged 0 to 11 years who had received one dose of the mumps vaccine, 50% were negative for mumps IgG antibodies. In the second group of children aged 12 to 18 years who had received two vaccine doses, the percentage of participants negative for mumps IgG antibodies decreased significantly. Conclusion: Our observations emphasise the need to conduct expanded sero-epidemiological investigations of the populations most affected by mumps and to immunise susceptible individuals.

  6. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  7. Sensitivity of some Immunoglobulin G class and subclass antibodies ...

    African Journals Online (AJOL)

    Indirect sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure serum antibody responses in onchocerciasis patients. Apparently, IgG antibody class was more sensitive than IgG1, IgG3 and IgG4 responses to Onchocerca volvulus adult worms sodium duodecyl sulphate (SDS) extracted crude ...

  8. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

    DEFF Research Database (Denmark)

    Tian, Xinsheng; Vestergaard, Bente; Thorolfsson, Matthias

    2015-01-01

    IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designe...... properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering....

  9. Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2017-02-01

    Full Text Available The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system. The study was conducted using a panel of characterized sera, as well as two reference ELISA sets of similar purpose. According to the results of ELISA informative value parameters, the ELISA we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered. In 4 out of 15 intralaboratory panel serum samples initially identified as negative, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant ELISA-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.

  10. Concurrent IgG4-related tubulointerstitial nephritis and IgG4 myeloperoxidase-anti-neutrophil cytoplasmic antibody positive crescentic glomerulonephritis: A case report.

    Science.gov (United States)

    Su, Tao; Yang, Li; Cui, Zhao; Wang, Su-Xia; Zhao, Ming-Hui

    2017-05-01

    IgG4-related disease (IgG4-RD) is a newly recognized systemic disease. The typical pathological finding in the kidney is abundant IgG4-positive plasma cell infiltration with characteristic storiform fibrosis in the interstitium. Antibodies of the IgG4 subclass have been linked to certain autoimmune diseases including antiproteinase 3 (PR3) anti-neutrophil cytoplasmic antibody (ANCA) of the IgG4 subclass. Here, we report a rare case of kidney injury with concurrent typical IgG4-related tubulointerstitial nephritis and IgG4 subclass of myeloperoxidase (MPO) ANCA-positive necrotizing crescentic glomerulonephritis. A 42-year-old Chinese man presented with repeated epigastric pain, sausage-shaped pancreas observed morphologically in computed tomography, effectiveness of prednisone therapy and was diagnosed with autoimmune pancreatitis. He subsequently developed acute kidney injury. The patient had an elevated serum IgG4, eosinophilia, and positive MPO-ANCA of IgG4-dominant subclass. Renal biopsy revealed necrotizing crescentic nephritis and typical IgG4-related tubulointerstitial nephritis. The patient was treated with a combination of corticosteroids and cyclophosphamide, and a course of rituximab was later added to deplete peripheral B cells. The patient responded well and his renal function improved. This is the first case report of an IgG4-RD with concurrent IgG4-related tubulointerstitial nephritis and IgG4 MPO-ANCA-associated necrotizing crescentic glomerulonephritis. It raises the difficulty in differentiation diagnosis of the two separate diseases that is worthy of further study.

  11. A three-layer immunoradiometric assay for antibodies in different immunoglobulin classes and its application to the detection of chicken thyroglobulin autoantibodies and of antibodies to sheep erythrocytes

    International Nuclear Information System (INIS)

    Carvalho, L.C.P. de; Roitt, I.M.; Wick, G.

    1980-01-01

    A versatile solid-phase assay for detection of antibodies in different immunoglobulin classes is described. The assay has been applied to: (1) the detection of IgG and IgM antithyroglobulin autoantibodies in chickens with spontaneous autoimmune thyroiditis, and (2) the detection of anti-sheep cell antibodies in normal chickens. Thyroglobulin-coated plastic tubes or formaldehyde-fixed sheep erythrocytes were used as the solid phase. Antisera were added in succession to the solid-phase antigen so as to form 3 antibody layers: (1) chicken antibody against the solid-phase antigen; (2) heavy-chain-specific rabbit anti-chicken immunoglobulin; and (3) 125 I-labelled goat anti-rabbit immunoglobulin. The assay is suitable for routine determinations on large numbers of samples; its sensitivity enables small volumes of serum to be tested and allows considerable economy in the use of valuable class-specific antisera. The radiolabelled reagent can be readily applied to other assays employing rabbit antisera. (Auth.)

  12. Quantification of anti-Leishmania antibodies in saliva of dogs.

    Science.gov (United States)

    Cantos-Barreda, Ana; Escribano, Damián; Bernal, Luis J; Cerón, José J; Martínez-Subiela, Silvia

    2017-08-15

    Detection of serum anti-Leishmania antibodies by quantitative or qualitative techniques has been the most used method to diagnose Canine Leishmaniosis (CanL). Nevertheless, saliva may represent an alternative to blood because it is easy to collect, painless and non-invasive in comparison with serum. In this study, two time-resolved immunofluorometric assays (TR-IFMAs) for quantification of anti-Leishmania IgG2 and IgA antibodies in saliva were developed and validated and their ability to distinguish Leishmania-seronegative from seropositive dogs was evaluated. The analytical study was performed by evaluation of assay precision, sensitivity and accuracy. In addition, serum from 48 dogs (21 Leishmania-seropositive and 27 Leishmania-seronegative) were analyzed by TR-IFMAs. The assays were precise, with an intra- and inter-assay coefficients of variation lower than 11%, and showed high level of accuracy, as determined by linearity under dilution (R 2 =0.99) and recovery tests (>88.60%). Anti-Leishmania IgG2 antibodies in saliva were significantly higher in the seropositive group compared with the seronegative (pLeishmania IgA antibodies between both groups were observed. Furthermore, TR-IFMA for quantification of anti-Leishmania IgG2 antibodies in saliva showed higher differences between seropositive and seronegative dogs than the commercial assay used in serum. In conclusion, TR-IFMAs developed may be used to quantify anti-Leishmania IgG2 and IgA antibodies in canine saliva with an adequate precision, analytical sensitivity and accuracy. Quantification of anti-Leishmania IgG2 antibodies in saliva could be potentially used to evaluate the humoral response in CanL. However, IgA in saliva seemed not to have diagnostic value for this disease. For future studies, it would be desirable to evaluate the ability of the IgG2 assay to detect dogs with subclinical disease or with low antibody titers in serum and also to study the antibodies behaviour in saliva during the

  13. Production and characterization of anti-human IgG F(ab')2 antibody fragment.

    Science.gov (United States)

    Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

    2018-04-10

    In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

  14. Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

    Science.gov (United States)

    Schlageter, A M; Kozel, T R

    1990-06-01

    A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.

  15. Indigenous Greenlanders have a higher sero-prevalence of IgG antibodies to Helicobacter pylori than Danes

    DEFF Research Database (Denmark)

    Milman, Nils; Byg, Keld-Erik; Andersen, Leif P

    2003-01-01

    To assess the sero-prevalence of IgG antibodies to Helicobacter pylori (H. pylori) in Greenlanders and compare with the sero-prevalence in Caucasian Danes.......To assess the sero-prevalence of IgG antibodies to Helicobacter pylori (H. pylori) in Greenlanders and compare with the sero-prevalence in Caucasian Danes....

  16. Presence of specific IgG antibody to grain dust does not go with respiratory symptoms.

    Science.gov (United States)

    Park, H S; Suh, C H; Nahm, D H; Kim, H Y

    1999-02-01

    A high prevalence of work-related symptoms in relation to grain dust exposure has been reported in grain dust workers, but the role of the specific IgG antibody is unknown. To study the possible role of specific IgG (sIgG) and specific IgG4 (sIgG4) in the development of work-related symptoms, sIgG and sIgG4 subclass antibodies against grain dust antigens were determined by ELISA in sera from 43 workers and 27 non-exposed controls. They were compared with results of specific IgE antibodies, exposure intensity and the presence of respiratory symptoms. SIgG and sIgG4 antibodies were detectable in almost all sera of exposed workers, and the prevalence were significantly higher than those of controls (pgrain dust exposure and may unlikely play a role in the etiology of respiratory symptoms.

  17. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    Science.gov (United States)

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection.

    Science.gov (United States)

    French, Martyn A; Abudulai, Laila N; Fernandez, Sonia

    2013-08-09

    The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of "protective" immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8⁺ T-cell responses restricted by "protective" HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  19. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

    Directory of Open Access Journals (Sweden)

    Sonia Fernandez

    2013-08-01

    Full Text Available The development of vaccines to treat and prevent human immunodeficiency virus (HIV infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK cell responses and plasmacytoid dendritic cell (pDC responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  20. Development of Two FhSAP2 Recombinant–Based Assays for Immunodiagnosis of Human Chronic Fascioliasis

    Science.gov (United States)

    Shin, Sun Hee; Hsu, Angel; Chastain, Holly M.; Cruz, Lorna A.; Elder, Eric S.; Sapp, Sarah G. H.; McAuliffe, Isabel; Espino, Ana M.; Handali, Sukwan

    2016-01-01

    In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)–FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG4, the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. PMID:27549636

  1. Development of Two FhSAP2 Recombinant-Based Assays for Immunodiagnosis of Human Chronic Fascioliasis.

    Science.gov (United States)

    Shin, Sun Hee; Hsu, Angel; Chastain, Holly M; Cruz, Lorna A; Elder, Eric S; Sapp, Sarah G H; McAuliffe, Isabel; Espino, Ana M; Handali, Sukwan

    2016-10-05

    In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)-FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG 4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG 4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG 4 , the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. © The American Society of Tropical Medicine and Hygiene.

  2. Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

    Directory of Open Access Journals (Sweden)

    Sabine Lederer

    Full Text Available In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV, Puumala (PUUV, Seoul (SEOV, Saaremaa (SAAV, Dobrava (DOBV, Sin Nombre (SNV or Andes (ANDV. For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97; SEOV (China, n=5; DOBV (Romania, n=7; SNV (Canada, n=23; ANDV (Argentina and Chile, n=52. The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96% and/or IgM (n=131; 72%, while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99% of the patient sera were IgG and 131 (71% IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%. Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%. Of the 89 control sera, 2 (2% showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV.

  3. The Induction of IgM and IgG Antibodies against HLA or MICA after Lung Transplantation

    OpenAIRE

    Paantjens, Annelieke W. M.; van de Graaf, Ed A.; Kwakkel-van Erp, Johanna M.; Hoefnagel, Tineke; van Ginkel, Walter G. J.; Fakhry, Farzia; van Kessel, Diana A.; van den Bosch, Jules M. M.; Otten, Henny G.

    2011-01-01

    The production of IgG HLA antibodies after lung transplantation (LTx) is considered to be a major risk factor for the development of chronic rejection, represented by the bronchiolitis obliterans syndrome (BOS). It has recently been observed that elevated levels of IgM HLA antibodies also correlates with the development of chronic rejection in heart and kidney transplantation. This study investigates the relationship between IgM and IgG antibodies against HLA and MICA after lung transplantati...

  4. Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing

    OpenAIRE

    Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

    1999-01-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTI...

  5. High levels of IgG4 antibodies to foods during infancy are associated with tolerance to corresponding foods later in life.

    Science.gov (United States)

    Tomicić, Sara; Norrman, Gunilla; Fälth-Magnusson, Karin; Jenmalm, Maria C; Devenney, Irene; Böttcher, Malin Fagerås

    2009-02-01

    Children with eczema and sensitization to foods are recommended skin care and, if food allergy is proven by challenge, an elimination diet. For most children the diet period is transient, but the process behind tolerance development and the influence of decreased allergen exposure is not fully known. The aim of the study was to investigate the effect of elimination diet on serum and salivary antibodies and to identify immunological parameters related to the ability to tolerate foods. Eighty-nine children, below 2 yr of age, with eczema and suspected food allergy were included. Recommended treatment was skin care to all children, and 60 children had a period of elimination diet. At 4(1/2) yr of age, the children were divided into two groups, based on if they had been able to introduce the eliminated foods, or not. Serum and salivary antibodies were analyzed with enzyme-linked immunosorbent assay and UniCAP before and after a 6-wk treatment period and at 4(1/2) yr of age. Children sensitized to egg and/or milk that could eat and drink the offending foods at 4(1/2) yr of age, had higher levels of Immunoglobulin G(4) antibodies to ovalbumin and beta-lactoglobulin and also higher IgG(4)/Immunoglobulin E ratios on inclusion in the study, than those who had to eliminate egg and/or milk from their diet, beyond 4(1/2) yr of age. The highest IgG(4)/IgE ratios were found in children with circulating IgE antibodies to egg and/or milk but negative skin prick test on inclusion. The 6-wk treatment period did not significantly affect the levels of serum and salivary antibodies. In conclusion, eczematous, food sensitized infants with high levels of IgG(4) and high ratios of IgG(4)/IgE antibodies to food allergens are more likely to consume these foods at 4(1/2) yr than infants with low levels and ratios.

  6. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

    1983-01-01

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  7. Antigen recognition by IgG4 antibodies in human trichinellosis

    Directory of Open Access Journals (Sweden)

    Pinelli E.

    2001-06-01

    Full Text Available The antibody isotype response to Trichinella spiralis excretory/secretory (ES products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 45 kDa component was strongly recognized by different antibody classes and subclasses. We observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

  8. Toxoplasma gondii IgG antibodies in HIV/AIDS patients attending ...

    African Journals Online (AJOL)

    Background: Toxoplasmosis is caused by Toxoplasma gondii, a parasite that gradually evolved to be the most opportunistic parasite that complicates the course of HIV/AIDS in developing countries. Aim: This study was undertaken to investigate the presence of Toxoplasma gondii IgG antibodies in HIVinfected patients ...

  9. An immunoenzymatic assay for the diagnosis of hepatitis A utilising immunoglobulin Y

    Directory of Open Access Journals (Sweden)

    Alexandre dos Santos da Silva

    2012-11-01

    Full Text Available The detection of anti-hepatitis A virus (HAV antibody levels by diagnostic kits in the convalescent period of disease generally use immunoglobulin G (IgG, which is expensive. An alternative to IgG is immunoglobulin Y (IgY, an immunoglobulin antibody encountered in birds and reptiles. The aim of this study was to develop a competitive immunoenzymatic assay to measure total anti-HAV antibody levels using anti-HAV IgY as the capture and conjugated immunoglobulins. For this purpose, anti-HAV IgY was conjugated to horseradish peroxidase (HRP and the optimal dilution of HRP-conjugated antibodies was evaluated to establish the competitive immuneenzymatic assay. The results obtained from our "in-house" assay were plotted on a receiver operator curve, which showed a sensitivity of 95% and a specificity of 98.8%, demonstrating that a competitive anti-HAV IgY immunoenzymatic assay developed "in house" could be used as an alternative to commercial assays that utilise IgG.

  10. Prevalence of rubella-specific IgG antibodies in unimmunized young female population

    Directory of Open Access Journals (Sweden)

    Jayakrishnan Thayyil

    2016-01-01

    Full Text Available Context: Rubella is a mild self-limiting disease all over the world; nevertheless, it is of significant public health importance due to its teratogenic effect of congenital rubella syndrome. Rubella vaccine is currently not included in the national immunization program in India. Rubella-specific IgG in the unvaccinated population is a marker of previous rubella infection. Rubella IgG estimation in children will provide data for initiation and necessary modification to the immunization strategy. Aims: In this background, this study was conducted with an aim to know the age-specific susceptibility of acquiring rubella infections and future risk of congenital rubella syndrome (CRS among girls. Settings and Design: This was a community-based, observational study. Participants and Methods: The study was conducted at a randomly selected rural area Mavoor Panchayath of Kozhikode District, Kerala, among adolescent girls. The estimation of rubella-specific IgG antibody was done by quantitative enzyme-linked immunosorbent assay method. IgG titer value of >15 IU was taken positive, 8-15 IU as equivocal, and <8 IU as negative. Statistical Analysis Used: Statistical analysis was performed using Statistical program for Social science version 16 for Windows. Chi-square test was applied to find out significant difference and Fisher′s exact test wherever applicable. Results: The data and blood sample collection was done from 250 girls. The mean IgG titer was 151.93 ± 128.78 IU, and as per the criteria, 68.3% were positive, 28.5% were negative, and 3.2% were equivocal. At this age, majority (68.3% of the girls get protection by natural infection without any vaccine. Some girls (32% may remain susceptible to infection during adulthood and pregnancy. Conclusions: Natural rubella infection was widely prevalent among child population and at this age. An immunization policy recommending rubella-containing vaccine is highly desirable to prevent rubella and CRS.

  11. A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments.

    Directory of Open Access Journals (Sweden)

    Hannah L Albritton

    Full Text Available Chlamydia trachomatis (CT is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs from two of the most common genital serovars (D and E were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined

  12. Monoclonal antibodies for use in an immunoradiometric assay for α-foetoprotein

    International Nuclear Information System (INIS)

    Hunter, W.M.; Bennie, J.G.

    1982-01-01

    The advantages offered by a mouse IgG 1 monoclonal antibody to human α-foetoprotein (AFP) for the preparation of [ 125 I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125 I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added [ 125 ]Ab was present in the sandwich. Linear response curves in the range 1-100 μg antigen/l incubate were obtained when [ 125 I]Ab was in slight excess. In this region an Ag : Ab ratio 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently 125 I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [ 125 I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system. (Auth.)

  13. Evaluation of Salivary Antibodies to Detect Infection with Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Mark B Loeb

    1997-01-01

    Full Text Available Helicobacter pylori infection is an important cause of peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of immunoglobulin (Ig G antibody. Salivary IgG antibody tests to detect H pylori infection offer a convenient and noninvasive method of diagnosis. To evaluate an IgG salivary antibody kit, saliva was collected from 157 out-patients with dyspepsia referred for endoscopy to a tertiary centre. A salivary IgG ELISA antibody assay was performed using the Helisal Helicobacter pylori (IgG assay kit, and at least four gastric biopsies were obtained. H pylori infection was confirmed by demonstration of the organism on Warthin-Starry silver stain (sensitivity 85%, specificity 55%. The prevalence of infection with H pylori was 30%. When the analysis was redone, excluding those treated with eradication therapy, the results were similar (sensitivity 86%, specificity 58%. The positive predictive value of the assay was 45% and the negative predictive value was 90%. Despite the ease of sampling, the assay used has limited diagnostic utility, lacking the predictive value to indicate which patients referred with dyspeptic symptoms to a tertiary care setting are infected with H pylori.

  14. Serum IgG antibodies from healthy subjects up to 100 years old react to JC polyomavirus.

    Science.gov (United States)

    Bononi, Ilaria; Mazzoni, Elisa; Pietrobon, Silvia; Manfrini, Marco; Torreggiani, Elena; Rossini, Marika; Lotito, Francesca; Guerra, Giovanni; Rizzo, Paola; Martini, Fernanda; Tognon, Mauro

    2018-08-01

    JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t-antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2-100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV-VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross-reactivity with the closely related SV40 and BKPyV polyomaviruses. © 2018 Wiley Periodicals, Inc.

  15. Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

    Science.gov (United States)

    Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

    2012-01-01

    The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

  16. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    International Nuclear Information System (INIS)

    Nordvall, S.L.; Uhlin, T.; Einarsson, R.

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated. (author)

  17. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Nordvall, S.L. (Dept. of Paediatrics, University Hospital, Uppsala, Sweden); Uhlin, T.; Einarsson, R. (Allergy Research, Pharmacia Diagnostics AB, Uppsala, Sweden)

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated.

  18. Evaluation of Immunity and Seropositivity of IgG Antibodies to Canine Parvoviruses in Vaccinated and Unvaccinated Dogs in Abeokuta, Nigeria.

    Science.gov (United States)

    Babalola, E T; Ijaopo, O K; Okonko, I O

    2016-01-01

    Canine Parvovirus (CPV) is a very contagious and virulent viral disease affecting domestic dogs all over the world causing high morbidity and mortality in dogs, especially puppies. This study aimed at determining the seropositivity of IgG antibodies against CPV in vaccinated and unvaccinated dogs and to evaluate the immune status of dogs presented in Abeokuta. Forty-eight dogs were enrolled in this study. These dogs were presented at random for treatment, routine checkup, and vaccination at the State Veterinary Hospital and Veterinary Teaching Hospital all in Abeokuta. All the dogs were fully maintained under domestic setting. Selection for study was done based on thorough examination of the dogs and their medical records. The clients were informed of the nature of the investigation. Blood samples were collected and analyzed for anti-CPV-IgG. In principle, protective immunity correlates with high antibody titers and this was determined using a commercially available immunocomb® test kit for anti-CPV IgG antibody. Of 48 dogs sampled, 38 (79.2%) had high level of anti-CPV antibody titer and 10 (20.8%) had low level of anti-CPV antibody titer. Twenty six (54.2%) were males while 22 (45.8%) were females. Forty-five (93.75%) dogs were exotic breeds while 3 (6.25%) dogs were mongrels. Thirty (62.5%) of the dogs were less than one year old and the age range of all dogs sampled was between 7 weeks and 7 years. There was no significant difference (P > 0.05) between sex and the level of immunity but significant differences (P canine parvovirus among dogs in Abeokuta, Nigeria. Of all variables evaluated, ages of dogs, breeds and post-vaccination period were the main correlates of the level of immunity to CPV. This study also showed agreement with previous studies in the diagnostic value of using the immunoblot ELISA assay for the rapid detection of anti-CPV IgG antibody.

  19. Detection of serum anti-sperm antibody in infertile couples with dot-immunogold filtration assay (DIGFA)

    International Nuclear Information System (INIS)

    Xie Xiaoxian

    2005-01-01

    Objective: To develop a new method for rapid detection of serum anti-sperm antibody in infertile couples. Methods: Human sperm antigen was prepared from pooled semen specimens of fertile males. Nitro-cellulose membrane was used as solid-phase carrier of the antigen. Colloidal gold pellet combined goat anti-human IgG was taken as labelled antibody. A dot-immunogold filtration assay system was established for test of serum anti-human sperm antibody. Serum specimens from 137 infertile couples were tested and the result compared with flat from ELISA. Results: The human sperm antigen would react with the anti-sperm antibody in the tested serum over the cellulose membrane through filtration and the result could be read with naked eye within 6 minutes. In this study of 137 infertile coupled, the anti-sperm antibody was positive in 21.9% of the female serum specimens and 13.19% of the males. Compared with the result from ELISA, the consistency rate was 96.1%. The sensitivity of the assay was 90.2% and specificity was 95.4%. The p reparation was stable after 6 months refrigerator storage. Conclusion: This newly developed DIGFA is very adequate for rap id detection of anti-sperm antibody and deserves popularization. (authors)

  20. IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

    Science.gov (United States)

    Koneczny, Inga; Stevens, Jo A A; De Rosa, Anna; Huda, Saif; Huijbers, Maartje G; Saxena, Abhishek; Maestri, Michelangelo; Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Tzartos, Socrates; Verschuuren, Jan; van der Maarel, Silvère M; van Damme, Philip; De Baets, Marc H; Molenaar, Peter C; Vincent, Angela; Ricciardi, Roberta; Martinez-Martinez, Pilar; Losen, Mario

    2017-02-01

    Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies

  1. Production of double antibody for radioimmunoassay (sheep anti-rabbit IgG antiserum)

    International Nuclear Information System (INIS)

    Silva, S.R. da.

    1993-01-01

    A second antibody (sheep anti-rabbit IgG antiserum) to be used in RIAs in which the first antibody is raised in rabbits was produced. For this production, initially the IgG was isolated from rabbit serum and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. Four sheep were immunized with 500 u g of purified rabbit IgG, emulsified in Freund Complete Adjuvant and administered by multisite subcutaneous injections. These injections were repeated at 20-days intervals and blood samples (40 ml) were taken from the jugular vein 10 days after the boosts for the evaluation of the antisera title. After each four boosts a great bleeding was done by the same route. Approximately 500 ml of serum were obtained in each bleeding per animal. The antisera were evaluated by the human thyrotropin RIA developed at IPEN laboratories employing reagents provided by NIDDKD, USA. These evaluations referred to the determination of the antisera title and of the ideal concentration of carrier IgG, to the study of the kinetic of precipitation and to the confirmation of the inexistent cross-reactivity with human IgG, in comparison with a reference antiserum of know precipitation characteristics supplied by the Radioassay System Laboratories. Approximately 3,6 l of antiserum (sheep anti-rabbit IgG serum) were produced from the four sheep, which presented title and precipitation characteristics very similar to those exhibited by the imported commercial product, even presenting higher titles. The results obtained in this work indicated that it was created enough experience for the production of this biological reagent for RIA, that could be done integrally in the country in greater scale, and at a very reduced cost. (author). 81 refs, 36 figs, 33 tabs

  2. Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

    Science.gov (United States)

    Tait, Brian D.

    2016-01-01

    This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

  3. A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide

    DEFF Research Database (Denmark)

    Barington, T; Sparholt, S; Juul, L

    1992-01-01

    A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...... antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality...... and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection...

  4. Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population.

    Science.gov (United States)

    Wong, Arthur; Lanyon, Janet M; McKee, Sara J; Linedale, Richard; Woolford, Lucy; Long, Trevor; Leggatt, Graham R

    2018-06-01

    Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. [Serum immunoglobulin IgG subclass distribution of antibody responses to pertussis toxin and filamentous hemagglutinin of Bordetella pertussis in patients with whooping cough].

    Science.gov (United States)

    Rastawicki, Waldemar; Smietańska, Karolina; Rokosz-Chudziak, Natalia; Jagielski, Marek

    2013-01-01

    The present study was aimed at determining the IgG subclass distribution against pertussis toxin (PT) and filamentous hemagglutinin (FHA) of Bordetella pertussis in patients with whooping cough. The total number of 222 serum samples obtained from patients suspected in clinical investigation for pertussis were tested separately by in-house ELISA for the presence of IgG antibodies to pertussis toxin and filamentous hemagglutinin. The percentage distribution of specific anti-PT and anti-FHA IgG subclass response was calculated only on the basis of group of sera confirmed in the present study as positive for total IgG antibodies (183 sera to PT antigen and 129 to FHA antigen). Paired serum specimens were obtained from 36 patients. Based on the results of determining the level of antibodies in the sera of 40 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus two standard deviations. Antibodies of IgG1 to pertussis toxin and filamentous hemagglutinin were diagnosed in 151 (82.5%) and 99 (76.7%), IgG2 in 72 (39.0%) and 50 (38.8%), IgG3 in 17 (9.3%) and 43 (33.3%), IgG4 in 55 (30.1%) and 53 (41.1%) serum samples, respectively. There were no significant differences in percentage of sera with IgG1, IgG2 and IgG3 in relation to age of the patients. However, the frequency of occurrence of IgG4 antibodies was highest in the group of the youngest children to the age of 6 years old (61.8% for PT and 68.0% for FHA), and decrease with age, reaching the minimum in the group of patients above 40 years old (13.2% and 4.2% for PT and FHA, respectively). We also found significantly higher frequency of IgG4 to PT and FHA antigens in men than in women. Statistically significant, essential changes in the pattern of IgG subclass during the course of infection were not found. In conclusion, this study showed that all four subclasses of IgG antibodies to pertussis toxin and filamentous hemagglutinin are produced during whooping cough.

  6. Specific IgG and its subclass antibodies after immunotherapy with gynandropsis gynandra

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    Latha G

    2005-01-01

    Full Text Available Background : About 10 to 15 % of the Indian population is known to suffer from major allergic disorders such as Asthma, Rhinitis, Atopic Dermatitis and Urticaria. Aeroallergens play a major role in the pathogenesis of respiratory allergic diseases. Among the aeroallergens, pollens are major causative agents. The predominance of pollen allergens necessitate the need to assess the specific immunotherapy (SIT in allergic patients. Objective : To evaluate the effect of immunotherapy based on the presence of IgG and its subclass antibodies towards whole pollen antigen of Gynandropsis gynandra (G.gynandra and its fractions. Material and Methods : A study was conducted in 30 bronchial asthma patients on immunotherapy, by assessing the levels of IgG and its subclasses specific to G. gynandra pollen. Results : There was a significant increase in IgG and its subclass antibodies to whole pollen antigen and its fractions i.e.> 90kD, 46-37kD and 36-32kD after the course of IT. Conclusion : The use of peptide fractions may be more appropriate instead of the whole pollen antigen to test the effect of immunotherapy.

  7. Quantitation of sperm bindable IgA and IgG in seminal fluid.

    Science.gov (United States)

    Howe, S E; Lynch, D M

    1986-05-01

    Seminal fluid and serum from 95 infertile males were assayed for sperm bindable immunoglobulins using an indirect ELISA with whole target sperm. The ELISA method was compared to seminal fluid and serum immobilization and agglutination assays (functional assays). In this infertile group, the ELISA assay was positive in 22% of seminal fluids (greater than 1.2 fg IgA/sperm and greater than 0.3 fg IgG/sperm). The seminal fluid antibodies were IgA and had an accompanying elevated IgG component in 78% of patients. There was a 96% correlation between negative seminal fluid functional assays and negative ELISA, and a 95% correlation between positive seminal fluid functional assays and positive ELISA. Positive serum sperm antibody tests were found in 71% of the infertile males with positive seminal fluid sperm antibodies, but 29% of the infertile males with strongly positive IgA seminal fluid sperm antibodies showed normal levels of serum sperm antibodies by either ELISA or functional assays. The ELISA method gives reproducible quantitation of sperm antibodies in seminal fluid and correlates well with accepted functional assays. Comparisons with serum sperm antibody assays suggests that seminal fluid sperm antibody analysis complements the serum analysis of sperm antibodies.

  8. Serum levels of IgG and IgG4 in Hashimoto thyroiditis.

    Science.gov (United States)

    Kawashima, Sachiko-Tsukamoto; Tagami, Tetsuya; Nakao, Kanako; Nanba, Kazutaka; Tamanaha, Tamiko; Usui, Takeshi; Naruse, Mitsuhide; Minamiguchi, Sachiko; Mori, Yusuke; Tsuji, Jun; Tanaka, Issei; Shimatsu, Akira

    2014-03-01

    Although IgG4-related disease is characterized by extensive infiltration of IgG4-positive plasma cells and lymphocytes of various organs, the details of this systemic disease are still unclear. We screened serum total IgG levels in the patients with Hashimoto thyroiditis (HT) to illustrate the prevalence of IgG4-related thyroiditis in HT. Twenty-four of 94 patients with HT (25.5%) had elevated serum IgG levels and their serum IgG4 was measured. Five of the 24 cases had more than 135 mg/dL of IgG4, which is the serum criterion of IgG4-related disease. One was a female patient who was initially treated as Graves' disease and rapidly developed a firm goiter and hypothyroidism. The biopsy of her thyroid gland revealed that follicular cells were atrophic with squamous metaplasia, replaced with fibrosis, which was compatible with the fibrous variant of HT. Immunohistochemical examination revealed diffuse infiltration of IgG4-positive plasma cells, and the serum IgG4 level was 179 mg/dL. The levels of IgG and IgG4 were positively correlated with the titers of anti-thyroglobulin antibody or anti-thyroid peroxidase antibody. In conclusion, at least a small portion of patients with HT with high titers of anti-thyroid antibodies may overlap the IgG4-related thyroiditis.

  9. Salivary IgG assay to detect Helicobacter pylori infection in an Indian adult population

    Directory of Open Access Journals (Sweden)

    Ramya Thirumala Krishnaswamy

    2012-01-01

    Conclusion: EIAgen H. pylori IgG assay is a noninvasive, moderately accurate, and sensitive method for the detection of H. pylori infection in saliva. Salivary anti H. pylori IgG test prior to endoscopy is a useful screening test for seroepidemiological studies.

  10. Evaluation of cysticercus-specific IgG (total and subclasses and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies Avaliação das respostas de anticorpos anti-cisticercos IgG (total e subclasses e IgE em amostras de líquido cefalorraquidiano de pacientes com neurocisticercose apresentando produção intratecal de anticorpos específicos IgG

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    2013-01-01

    Full Text Available In the present study, an enzyme-linked immunosorbent assay (ELISA standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses and IgE antibodies in cerebrospinal fluid (CSF samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA=1.17 and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49 and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46 and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12 and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85 and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60 and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.No presente estudo, uma reação imunoenzimática (ELISA padronizada com o fluido vesicular de cisticercos de Taenia solium foi utilizada para avaliar as respostas de anticorpos anti-cisticercos IgG (total e subclasses e IgE em amostras de líquido cefalorraquidiano (LCR de pacientes com neurocisticercose apresentando produção intratecal de anticorpos específicos IgG e pacientes com outras desordens neurológicas. Os seguintes resultados foram obtidos: ELISA-IgG: 100% de sensibilidade (mediana das absorbâncias das reações ELISA (MAE=1,17 e especificidade 100%; ELISA-IgG1: sensibilidade 72,7% (MAE=0,49 e especificidade 100%; ELISA-IgG2

  11. Prodduction of clone secretor of antibodies (IgG againt of infection bursal disease virus

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    Sandra Yuliet Marín

    2015-06-01

    Full Text Available ABSTRACT. Marín S.Y., dos Santos B.M., Patarroyo J.H. & Vargas M.I. [Prodduction of clone secretor of antibodies (IgG againt of infection bursal disease virus.] Produção de clones secretores de anticorpos (IgG contra o vírus da doença infecciosa bursal. Revista Brasileira de Medicina Veterinária, 37(2:238-144, 2015. Programa de Pós-Graduação em Medicina Veterinária, Universidade Federal de Viçosa, Avenida PH Rolfs, s/n, Viçosa, MG 36570- 000, Brasil. E-mail: bmsantos@ufv.br Three clones secreting of antibodies (Abs IgG against infection bursal disease virus IBDV was development. The IBDV was strain S706 (the intermediate vaccine was replicated in VERO cell and purified by sucrose gradient, for ELISA and mice inoculation. For the immunization of the mice BALB/c using as a saponin adjuvant, that allowed an inflammation reaction which enhanced the antibody response, detectable by ELISA. The fusion of splenic cells of the immunized mice and the mieloma SP2/0 resulted in 2 hybridoma families (2H11 and 5C7. After cloning by limiting dilution, 3 clones secretors of Abs from IgG class were obtained. The 3 obtained Abs were capable to reveal the proteins turn VPX and VP2 by “western blotting”, respectively of 47 kDa and 41 kDa. The definition of the isotypes recognized by obtained Abs must be object of characterization to allow the use of the antibodies in immunodiagnostic tests such as immunofluorescence, immunocitochemistry or capture ELISA, for epidemiologic of the disease researches or to differentiate vaccine’s virus of the field virus.

  12. Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and "bispecific" monoclonal antibody formation.

    Science.gov (United States)

    Debaene, François; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

    2013-10-15

    Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

  13. An immunochemical method for the quantitation of insulin antibodies

    International Nuclear Information System (INIS)

    Reeves, W.G.; Kelly, U.

    1980-01-01

    A 125 I-labelled insulin binding assay is described in which IgG antibody is precipitated by the addition of an optimal concentration of second antibody. Other features include the removal of unlabelled insulin from test sera prior to assay and the use of 22 Na as a volume marker. This approach overcomes problems associated with previous assays for insulin antibodies. Clear differences are seen in the IgG insulin binding capacity (IBC) of sera from patients with insulin resistance and injection site lipo-atrophy when compared with insulin-treated diabetics who lack such complications. The precision and flexibility of this technique make it particularly suitable for studies of the immune response to different species and forms of insulin. (Auth.)

  14. Human IgG1 antibodies suppress angiogenesis in a target-independent manner

    NARCIS (Netherlands)

    Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

    2016-01-01

    Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in

  15. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    Science.gov (United States)

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  16. Anti-CagA IgG Antibody is Independent from Helicobacter pylori vacA and cagA Genotypes

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    Hashem Fakhre Yaseri

    2015-12-01

    Full Text Available Background: Helicobacter pylori strains have two classical virulence genes, the cytotoxinassociated A (cagA gene and the vacuolating cytotoxin A (vacA gene, which are located in thecag pathogenicity island (cagPAI. Serum immunoglobulin G (IgG antibodies to H. pylori,especially, the CagA antigen may be a reliable marker for selection of dyspeptic patients for upperendoscopy.Methods: Serum sample of 129 dyspeptic patients with positive H. pylori, were tested for serumIgG Anti-CagA antibody by ELISA. The presence of the cagA and vacA genotypes weredetermined using polymerase chain reaction (PCR on biopsy samples taken via endoscopy.Results: Positive serum IgG anti-CagA antibodies in patients with cagA+/vacA+ and cagA+/vacA- genotypes were 22/23 (95.6% and 18/19 (94.7%, respectively. In addition, serum IgG anti-CagAantibodies in patients with cagA-/vacA+ and cagA-/vacA- genotypes were 22/47 (46.8% and 33/40(82.5%, respectively.Conclusions: It can be concluded that the serum IgG anti-CagA antibody alone could selectpatients with dyspepsia following upper endoscopy. The assessment of vacuolating cytotoxinactivity of H. Pylori is, therefore, not required, even when vacA gene is positive. This hypothesisneeds to be studied in a large number of patients with dyspepsia.

  17. Hormesis of specific IgG antibody to rabies virus in serum of mice irradiated with low dose γ-rays

    International Nuclear Information System (INIS)

    Liu Qingjie; Chen Deqing

    1998-01-01

    Objective: To explore the effect of low dose ionizing radiation on specific antibody in mouse serum. Methods: Kunming strain male mice, weighing 18-22 g, aged 6-8 weeks, were immunized intraperitoneally with rabies vaccine after exposure to cobalt-60 γ-rays. The specific IgG antibody against rabies virus in mouse serum was measured. Results: (1) The serum levels of specific IgG in mice irradiated with 5-30 cGy γ-rays were significantly elevated in comparison with those in control mice (P<0.01), the optimum stimulating dose being 10 cGy. (2) Exposure to 10 cGy caused significant enhancement and earlier emergence of the peak level of specific IgG in serum. (3) The hormesis of specific IgG to rabies virus induced by 10 cGy γ-rays could last one week at least. Conclusion: Low dose ionizing radiation can enhance the level of specific antibody in mouse serum, and this effect can last for one week at least

  18. Depigmented allergoids reveal new epitopes with capacity to induce IgG blocking antibodies.

    Science.gov (United States)

    López-Matas, M Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

    2013-01-01

    The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.

  19. Evaluation of Antigen-Specific IgM and IgG Production during an In Vitro Peripheral Blood Mononuclear Cell Culture Assay

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    Yoshiko Matsuda

    2017-07-01

    Full Text Available The recent attention given to diseases associated with memory B-cell (mBC-produced antibodies (Abs suggests the need for a similar in vitro assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs with the intent to collect mBC-derived Abs in vitro and maintain their cell–cell contact-dependent interactions with helper T-cells. PBMCs were cultured with interleukin (IL-21, CpG-oligodeoxynucleotides (ODN, phorbol myristate acetate (PMA, and phytohemagglutinin/leucoagglutinin (PHA-L in 24-well flat-bottom plates (5 × 105 cells/well. A culture supernatant analysis of PBMCs from healthy donors (n = 10 indicated that antigen-specific IgM Ab levels in a PBMC culture supernatant might be better able to demonstrate the antigen sensitization status in a smaller peripheral blood sample, compared to IgG because Epstein–Barr virus-specific IgM mBCs circulate peripherally at a significantly higher frequency once antiviral humoral immunity has stabilized. Thus, our in vitro assay demonstrated the potential significance of antigen-specific IgM Ab production in the culture supernatants. Furthermore, an analysis of cultured PBMCs from allograft kidney recipients (n = 16 sensitized with de novo donor-specific human leukocyte antigen (HLA-specific Abs (DSAs showed that IgM-type HLA-specific Abs were detected mainly from the culture supernatants from PBMCs of patients with stable graft function, whereas IgG isotype HLA Abs were detectable only from patients with biopsy-proven antibody-mediated rejection. In other words, these IgG isotype Abs also represented an activated humoral immune response in vivo. Additionally, IgM- and IgG-expressing mBCs from healthy donors (n = 5 were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19+ B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 105 cells/well, and the resulting in vitro analysis provided some

  20. Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

    International Nuclear Information System (INIS)

    Benkirane, M.; Bon, D.; Bellot, F.; Prince, P.; Delori, P.; Hassoun, J.; Carayon, P.

    1987-01-01

    Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10 9 -10 11 mol -1 .l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or αhCG. Four reacted with these hormones and recognized the α subunit of hCG. One cross-reacted only with HFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-βTSH) and another (anti-α) labelled with 125 I. This assay was highly specific and demonstrated a sensitivity level of 0.1 μIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections. 20 refs.; 6 figs.; 2 tabs

  1. Human anti-rhesus D IgG1 antibody produced in transgenic plants

    DEFF Research Database (Denmark)

    Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed

    2002-01-01

    antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent......Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...... properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs....

  2. Diagnostic accuracy and comparison of two assays for Borrelia-specific IgG and IgM antibodies

    DEFF Research Database (Denmark)

    Dessau, Ram

    2013-01-01

    characteristic (ROC) curves to optimize and standardize test interpretation, it was shown that testing with both IDEIA IgG and IgM was comparable to testing with Liaison IgG alone by comparing the area under the curve of the diagnostically relevant 25 % partial ROC curve (P = 0.1). When using the Liaison Osp......M combined) were 85 and 95 % and for the Liaison (VlsE IgG) method were 67 and 96 %, respectively. Methods for test evaluation, test interpretation and statistical testing are presented and discussed. In conclusion, Liaison VlsE IgG alone and IDEIA IgG/IgM combined showed a high and comparable discriminatory...

  3. Primary cytomegalovirus infection in pregnant Egyptian women confirmed by cytomegalovirus IgG avidity testing.

    Science.gov (United States)

    Kamel, N; Metwally, L; Gomaa, N; Sayed Ahmed, W A; Lotfi, M; Younis, S

    2014-01-01

    To determine the frequency of primary cytomegalovirus (CMV) infection in pregnant Egyptian women using CMV IgG avidity testing. A cross-sectional study was conducted at Suez Canal University Hospital, Ismailia, Egypt. A total of 546 pregnant women, presenting for routine antenatal screening, were tested for CMV IgG and IgM using a commercially available enzyme-linked immunosorbent assay (ELISA). Sera from CMV IgM-positive women were tested by CMV IgG avidity assay. All the 546 pregnant women were seropositive for anti-CMV IgG. Of the 546 women, 40 (7.3%) were positive or equivocal for IgM antibodies. All sera from the 40 women (IgG+/IgM+) showed a high or intermediate CMV IgG avidity index. Of the 40 women, 23 (57.5%) were in the second or third trimesters of pregnancy and had their first-trimester blood retrieved, and the tested CMV IgG avidity assay showed a high avidity index. Women who were IgM positive had no primary CMV infection in the index pregnancy as evidenced by the high CMV IgG avidity testing. © 2013 S. Karger AG, Basel.

  4. Detection of anti neutrophil antibodies by radio-iodinated protein A binding test

    International Nuclear Information System (INIS)

    Cartron, J.; Muller, J.Y.; Tchernia, G.; Paule, B.; Varet, B.

    1983-01-01

    The granulocyte associated IgG in normal and neutropenic subjects has been determined by a direct quantitative assay using radiolabeled staphylococcal protein A. This assay allows to postulate an immunological mechanism to explain the neutropenia in 19 cases of neutropenias associated with malfunctions of the immune system and in 4 cases of idiopathic neutropenias. Discussed in this report is the possible interaction of immune complexes bound in vivo to the granulocytes. By an immunofluorescence test, it has been possible to detect IgG or IgM antibodies in only 52% of patients with a positive direct assay. The determination of granulocyte-associated IgG is therefore a better indicator for defining an auto-immune neutropenia than the detection of free antibodies in the sera [fr

  5. Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

    International Nuclear Information System (INIS)

    Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

    1985-01-01

    Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr 51 Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not

  6. Pneumococcal polysaccharide vaccination elicits IgG anti-AB blood group antibodies in healthy individuals and patients with Type I diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Wendelin Wolfram

    2016-11-01

    Full Text Available Hypothesis: Blood group antibodies are natural antibodies that develop early in life in response to cross-reactive environmental antigens in the absence of antigen encounter. Even later in life structural similarities in saccharide composition between environmental antigens such as bacterial polysaccharides and blood group A/B antigens could lead to changes in serum levels, IgM/IgG isotype and affinity maturation of blood group anti-A/B antibodies. We adressed the question whether immunization with pneumococcal polysaccharide (PnP vaccine (PPV Pneumovax®23 could have such an effect in patients with with type I diabetes mellitus (DM I, an autoimmune disease where an aberrant immune response to microbial antigens likely plays a role.Methods: Anti-PnP IgM and IgG responses were determined by ELISA and the Diamed-ID Micro Typing System was used to screen anti-A/B antibody titer before and after Pneumovax®23 immunization in 28 healthy individuals and 16 patients with DM I. In addition, surface plasmon resonance (SPR technology using the Biacore® device and a synthetic blood group A/B trisaccharide as the antigen was applied to investigate IgM and IgG anti-A/B antibodies and to measure antibody binding dynamics. Results: All healthy individuals and DM I patients responded with anti-PnP IgM and IgG antibody production four to six weeks after Pneumovax®23 (Pn23 immunization, while no increase in blood group anti-A/B antibody titer was observed when measured by the Diamed-ID Micro Typing System. Interestingly, isotype-specific testing by SPR-technology revealed an increase in blood group anti-A/B IgG, but not IgM, following Pn23 immunization in both patients and controls. No change in binding characteristics of blood group anti-A/B antibodies could be detected following Pn23 vaccination, supporting the assumption of an increase in IgG antibody titer with no or very little affinity maturation.Conclusion: The study provides evidence for epitope sharing

  7. Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

    Directory of Open Access Journals (Sweden)

    M. Angeles López-Matas

    2013-01-01

    Full Text Available Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.

  8. A meta-analysis of the prevalence of toxoplasma gondii igg antibodies in patients with mental disorders

    Directory of Open Access Journals (Sweden)

    Angela Dragomir1,

    2016-12-01

    Full Text Available OBJECTIVES AND BACKGROUND Toxoplasma gondii infection has been recently associated with schizophrenia and other psychiatric disorders. The aim of the present study was to evaluate the prevalence of T. gondii infection in psychiatric patients by using meta-analytical methods. MATERIALS AND METHODS By systematic research of PUBMED Database, we identified several articles on this issue. We included casecontrol studies focused on the seroprevalence of T. gondii (IgG antibodies in patients with psychiatric disorders and healthy controls, published over the past 10 years R3.2.2. free software for statistical computing and graphics was used to perform the meta-analysis. Data were pooled using a random effects model and Mantel-Haenszel method. RESULTS The PUBMED Database showed references to only seven scientific papers that investigated the prevalence of T. gondii IgG antibodies in psychiatric patients. Six hundred seventy three patients and seven hundred seventy four controls coped with the inclusion criteria and were used in our analysis. We found a significant increase of T. gondii IgG antibodies in patients with schizophrenia and other psychiatric disorders compared with controls (41.6% vs 24.54%, OR = 2.16, 95% CI = [1.45-3.21], P = .001. CONCLUSIONS An increased seroprevalence of T. gondii IgG antibodies has been reported in psychiatric patients. Our study suggests that T. gondii infection may be relevant in order to determine and understand the complex etiology of schizophrenia and other psychiatric disorders. Graphical abstract: Meta-analysis of the prevalence of T. gondii IgG antibodies in patients with mental disorders REFERENCES 1. Hamidinejat H, Ghorbanpoor M, Hosseini H, Alavi SM, Nabavi L, Jalali MH, Borojeni MP, Jafari H, Mohammadaligol S. Toxoplasma gondii infection in first-episode and inpatient individuals with schizophrenia. Int J Infect Dis. 2010;14:e978-81. doi: 10.1016/j.ijid.2010.05.018. 2. Alvarado-Esquivel C, Carrillo-Oropeza D

  9. Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Meurman, O.; Terho, P.; Sonck, C.E.

    1981-01-01

    A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement.

  10. Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Meurman, O.; Terho, P.; Sonck, C.E.

    1981-01-01

    A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement. (orig.)

  11. Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

    OpenAIRE

    L?pez-Matas, M. Angeles; Gallego, Mayte; Iraola, V?ctor; Robinson, Douglas; Carn?s, Jer?nimo

    2013-01-01

    Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtain...

  12. Amylolytic activity of IgM and IgG antibodies from patients with multiple sclerosis.

    Science.gov (United States)

    Saveliev, Andrew N; Ivanen, Dina R; Kulminskaya, Anna A; Ershova, Nadezhda A; Kanyshkova, Tat'yana G; Buneva, Valentina N; Mogelnitskii, Alexander S; Doronin, Boris M; Favorova, Olga O; Nevinsky, Georgy A; Neustroev, Kirill N

    2003-05-01

    IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.

  13. Radioimmunoassay of class-specific antibodies (RIACA): chicken antibodies to DNP

    International Nuclear Information System (INIS)

    Viljanen, M.K.; Granfors, K.; Toivanen, P.

    1977-01-01

    A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 125 I-labelled anti-chicken-μ or anti-chicken-γ. The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes it possible to quantitate class-specific antibodies on weight units

  14. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    Science.gov (United States)

    Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  15. Expression of human FcgammaRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding.

    Science.gov (United States)

    Armour, Kathryn L; Smith, Cheryl S; Clark, Michael R

    2010-03-31

    The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3

  16. Preferential decrease in IgG4 anti-citrullinated protein antibodies during treatment with tumour necrosis factor blocking agents in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Bos, W. H.; Bartelds, G. M.; Vis, M.; van der Horst, A. R.; Wolbink, G. J.; van de Stadt, R. J.; van Schaardenburg, D.; Dijkmans, B. A. C.; Lems, W. F.; Nurmohamed, M. T.; Aarden, L.; Hamann, D.

    2009-01-01

    To investigate the dynamics of IgG1 and IgG4 anti-citrullinated protein antibody (ACPA) subclasses during anti-tumour necrosis factor (TNF) treatment in patients with rheumatoid arthritis (RA). IgG, IgG1 and IgG4 ACPA levels were determined by ELISA on anti-citrullinated fibrinogen (ACF) and IgG1 :

  17. Anti-Taenia solium metacestode IgG antibodies in serum samples from inhabitants of a central-western region of Brazil

    Directory of Open Access Journals (Sweden)

    Oliveira Heliana B. de

    2006-01-01

    Full Text Available A total of 354 serum samples from inhabitants who frequent the Clinical Laboratory in Catalão, Goiás, in the central-western region of Brazil, were collected from June to August, 2002. The samples were evaluated by indirect immunofluorescence antibody tests and an enzyme linked immunosorbent assay in order to detect anti-Taenia solium metacestode IgG antibodies. Reactive and inconclusive samples were tested by Western blotting (WB. Considering WB as a confirmation, the frequency of antibodies in the serum samples of the above population was 11.3% (CI 5.09 - 17.51. The immunodominant bands most frequently recognized in WB were 64-68 kDa (97.5% and 47-52 kDa (80%. The percentage of seropositivity to cysticercosis was significantly higher for individuals residing in areas without sewage systems (p < 0.0001. In conclusion, the results indicate a probable endemic situation of cysticercosis in this population. These results reinforce the urgent need for control and prevention measures to be taken by the local public health services.

  18. Correlation between the Amount of Anti-D Antibodies and IgG Subclasses with Severity of Haemolytic Disease of Foetus and Newborn

    Directory of Open Access Journals (Sweden)

    Emilija Velkova

    2015-05-01

    CONCLUSIONS: The titers of the pregnant women serum those are lower than 32 and those higher than 1000 can well predict HDFN. The titers of anti-D antibodies between 64 and 512 have no exact predictive value. IgG1 and IgG3 subclasses of anti-D have no predictive value by themselves, and cannot foresee the outcome of HDFN. The research study results suggest that IgG1 and IgG3 should be included in a multi – parameter protocol for evaluation of the HDFN intensity. They can give a real assessment of the expected HDFN intensity in combination with the titer hight and the significance of the antibodies.

  19. Preferential decrease in IgG4 anti-citrullinated protein antibodies during treatment with tumour necrosis factor blocking agents in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Bos, W.H.; Bartelds, G.M.; Vis, M.; van der Horst, A.R.; Wolbink, G.J.; van de Stadt, R.J.; van Schaardenburg, D.; Dijkmans, B.A.C.; Lems, W.F.; Nurmohamed, M.T.; Aarden, L.; Hamann, D.

    2009-01-01

    Objective: To investigate the dynamics of IgG1 and IgG4 anti-citrullinated protein antibody ( ACPA) subclasses during anti-tumour necrosis factor (TNF) treatment in patients with rheumatoid arthritis ( RA). Methods: IgG, IgG1 and IgG4 ACPA levels were determined by ELISA on anti-citrullinated

  20. Preferential decrease in IgG4 anti-citrullinated protein antibodies during treatment with tumour necrosis factor blocking agents in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Bos, W.H.; Bartelds, G.M.; Vis, M.; Horst, A.; Wolbink, G.; van de Stadt, R.J.; van Schaardenburg, D.; Dijkmans, B.A.C.; Lems, W.F.; Nurmohamed, M.T.; Aarden, L.; Hamann, D.

    2009-01-01

    Objective: To investigate the dynamics of IgG1 and IgG4 anti-citrullinated protein antibody (ACPA) subclasses during anti-tumour necrosis factor (TNF) treatment in patients with rheumatoid arthritis (RA). Methods: IgG, IgG1 and IgG4 ACPA levels were determined by ELISA on anti-citrullinated

  1. Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

    Science.gov (United States)

    Broger, Tobias; Basu Roy, Robindra; Filomena, Angela; Greef, Charles H; Rimmele, Stefanie; Havumaki, Joshua; Danks, David; Schneiderhan-Marra, Nicole; Gray, Christen M; Singh, Mahavir; Rosenkrands, Ida; Andersen, Peter; Husar, Gregory M; Joos, Thomas O; Gennaro, Maria L; Lochhead, Michael J; Denkinger, Claudia M; Perkins, Mark D

    2017-04-01

    Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  2. IgG4 breaking the rules

    NARCIS (Netherlands)

    Aalberse, Rob C.; Schuurman, Janine

    2002-01-01

    Immunoglobulin G4 (IgG4) antibodies have been known for some time to be functionally monovalent. Recently, the structural basis for this monovalency has been elucidated: the in vivo exchange of IgG half-molecules (one H-plus one L-chain) among IgG4. This process results in bispecific antibodies that

  3. Genus and species-specific IgG and IgM antibodies pulmonary tuberculosis

    International Nuclear Information System (INIS)

    Butt, T.; Abbassi, S.A.; Ahmad, R.N.; Mahmood, A.; Karamat, K.A; Malik, H.S.; Anwar, M.

    2004-01-01

    Objective: To evaluate three different enzyme immunoassays for serological diagnosis of pulmonary tuberculosis and to compare their diagnostic accuracy in different combinations. Subjects and Methods: Sera from patients suffering from pulmonary tuberculosis (n=94) with sputum positive for acid fast bacilli (AFB) and sera from control group of healthy individuals (n=90) with sputum negative for AFB were tested by Pathozyme-Myco G EIA, Pathozyme-TB Complex Plus EIA and Pathozyme Myco M EIA kits for the genus-specific IgG and IgM, and the species-specific IgG antibodies against antigens of Mycobacterium tuberculosis. Results: The detection of IgG against genus-specific antigens by Pathozyme-Myco G had a sensitivity of 46% and a specificity of 93%, of IgG against species-specific antigens by Pathozyme- TB Complex Plus had a sensitivity of 64% and specificity of 97% and of IgM against genus-specific antigens by Pathozyme Myco M had a sensitivity of 67% and specificity of 98%. When the results of these immunoassays were evaluated in combination, their sensitivity improved. Combination of genus-specific IgM and species-specific IgG yielded best results with a sensitivity of 87% and specificity of 93%. Conclusion: The sensitivity of serological diagnosis of tuberculosis is low, but it can be increased by utilizing a combination of several antigens. (author)

  4. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    International Nuclear Information System (INIS)

    Ryu, T.; Davis, J.M.; Schwartz, K.A.

    1990-01-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding

  5. Human IgG4: a structural perspective.

    Science.gov (United States)

    Davies, Anna M; Sutton, Brian J

    2015-11-01

    IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs. © 2015 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

  6. Detection of IgM and IgG antibodies to Chlamydophila pneumoniae in pediatric community-acquired lower respiratory tract infections

    Directory of Open Access Journals (Sweden)

    Surinder Kumar

    2011-01-01

    Full Text Available Context: Chlamydophila pneumoniae (C. pneumoniae is an emerging infectious agent with a spectrum of clinical manifestations including lower and upper respiratory tract infections. Aims: To investigate the role of C. pneumoniae in community-acquired lower respiratory tract infections (LRTIs in children using serological tests. Settings and Design: Two hundred children, age 2 months to 12 years, hospitalized for community-acquired LRTIs were investigated for C. pneumoniae etiology. Materials and Methods: We investigated 200 children hospitalized for community-acquired LRTIs, using ELISA for detecting anti-C. pneumoniae IgM and IgG antibodies. The demographic, clinical and radiological findings for C. pneumoniae antibody positive and C. pneumoniae antibody negative cases were compared. Statistical Analysis Used: Data analysis was performed by Chi-square test and Fisher′s exact tests using Epi Info (2002. Results: Clinical and radiological findings in both the groups were comparable. Serological evidence of C. pneumoniae infection was observed in 12 (6% patients; specific IgM antibodies were detected in 11 (91.67%; specific IgG antibodies in 1 (8.33% patients, while 4-fold rise in C. pneumoniae IgG antibody titers were noted in none of the patients. Conclusions: C. pneumoniae has a role in community-acquired LRTIs, even in children aged < 5 years. Serological detection using ELISA would enable pediatricians in better management of C. pneumoniae infections.

  7. Isolation and purification of rabbit imunoglobulin (IgG) for the production of a second antibody for radioimunoassay

    International Nuclear Information System (INIS)

    Silva, S.R. da; Borghi, V.C.

    1990-03-01

    Immunoglobulin (IgG) from rabbit serum was isolated and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. The efficiency of the procedure was followed by total protein determination during all purification steps. The purity of the final product was verified through immunoelectroforesis of IgG with sheep serum anti-rabbit whole serum. Were obtained 850 mg of pure IgG, enough for the immunization of several sheeps to be used in the production of a second antibody for radioimmunoassay. (author) [pt

  8. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

    DEFF Research Database (Denmark)

    de Jong, R. N.; Beurskens, F. J.; Verploegen, S.

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

  9. [Determination of cytomegalovirus IgG synthesis by the albumin correction in the aqueous humor of posner-schlossmann syndrome].

    Science.gov (United States)

    Wang, X L; Wang, Z J; Tang, L; Cao, W W; Sun, X H

    2017-02-11

    Objective: To evaluate the usefulness of albumin correction in determination of cytomegalovirus IgG in the aqueous humor of Posner-Schlossman syndrome (PSS) patients. Methods: Cases series studies. Forty-two patients (26 men and 16 women) who were diagnosed as PSS were enrolled from Oct. 2009 to Oct. 2015 at the Eye and ENT Hospital. During the same period, 20 patients with primary open-angle glaucoma (POAG) and 30 patients with bacterial endophthalmitis or retinal necrosis were enrolled as negative control group and inflammatory disease control group, respectively. Aqueous humor and serum samples were assayed to detect CMV IgG by enzyme-linked immunosorbent assay (ELISA), and albumin by scattering immunonephelometry. CMV DNA in aqueous humor was assayed by polymerase chain reaction (PCR). The ratio which was calculated as the (aqueous humor CMV IgG/serum CMV IgG)/(aqueous humor concentration of albumin/serum albumin concentration) over 0.6 was considered as intraocular antibody formation. Performance of differentiating control eyes from eyes with CMV-positive PSS was evaluated by the receiver operating characteristic curve. The ANOVA test, Mann-Whitney test and Chi-square test were performed to compare the differences among groups. Results: The detectable rate of CMV IgG antibody in the aqueous humor was 76.2%, 100.0% and 10.0% in PSS, inflammatory disease control and POAG groups, respectively. The levels of CMV IgG antibody in the PSS groups were significantly higher than that of POAG groups ( Z= 4.23, Palbumin was 71.4%, 3.3% and 0.0%.The corrected positive rate in PSS groups was significantly higher than that of the inflammatory disease control and POAG groups (χ(2)=30.38, Palbumin could effectively improve aqueous antibody specificity in PSS patients. Furthermore, CMV IgG antibody ratio combined with PCR could improve the sensitivity of CMV detection. All of which help clarify the CMV infection in PSS in CMV DNA negative eyes. (Chin J Ophthalmol, 2017, 53

  10. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  11. [Effect comparison between two ELISA kits in IgG antibody detection of Echinococcus granulosus].

    Science.gov (United States)

    Chu, Yan-Hong; Cai, Yu-Chun; Ai, Lin; Lu, Yan; Zhang, Jia; Chen, Jia-Xu

    2013-06-01

    To compare the effects of two ELISA kits on IgG antibody detection of human Echinococcus granulosus. A Total of 134 sera of patients with echinococcosis, paragonimiasis westermani, clonorchiasis sinensis, schistosomiasis japonica, and cysticercosis cellulosae, and normal persons were detected by two IgG ELISA kits produced by different companies. Furthermore, the specificity, sensitivity and cross reactivity were counted and analyzed statistically. The sensitivity and specificity were extremely high of the two kits as 100.00%. The cross-reactivity rates were 25.00% (paragonimiasis westermani), 26.09% (clonorchiasis sinensis), 10.00% (schistosomiasis japonica), and 87.5% (cysticercosis), respectively, by using the kit produced by the Combined Company in Shenzhen; the cross-reactivity rates were 5.00% (paragonimiasis westermani), 13.04% (clonorchiasis sinensis), 20.00% (schistosomiasis japonica), and 93.75% (cysticercosis) respectively, by using the kit produced by Haitai Company in Zhuhai. In addition, there was a significant difference of Paragonimus westermani detection (P 0.05) between the two kits. Both ELISA kits on IgG antibody detection of human Echinococcus granulosus have the advantages of a high sensitivity, specificity, convenience and high-speed. However, it is also in urgent need to further solve the cross-reactivity of Echinococcus granulosus with other parasites, in order to improve the accuracy of early diagnosis.

  12. AN EVALUATION STUDY OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA USING RECOMBINANT PROTEIN GRA1 FOR DETECTION OF IGG ANTIBODIES AGAINTS TOXOPLASMA GONDII INFECTIONS

    Directory of Open Access Journals (Sweden)

    Nina Difla Muflikhah

    2017-08-01

    Full Text Available Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo endothelial and parenchymal cells of human and animals (mammals and birds. Some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. Its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. Early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. Enzyme-linked immunosorbent assay (ELISA allow the testing of a large number samples within short time frame and based on antibody or antigen detection. This study aimed to know the sensitivity and specificity of recombinat protein GRA1 as antigen using ELISA methods. We tested the sensitivity and spesificity of GRA1 protein as antigen in ELISA methods to diagnose toxoplasmosis and compared with ELISA Kit Commercial. Reliable laboratory testing is important to detect Toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. Seventy sera collected and tested using both indirect ELISA, commercial ELISA kit and GRA1 protein coated as antigen. Fourty eight and fifty one samples showed positive IgG antibody result of ELISA-GRA1 and ELISA kit. Negative sample tested by ELISA-GRA1 was 22 samples and 19 sample tested by ELISA Kit. The sensitivity and specificity of GRA1-based on ELISA were 100% and 86.36%, positive prediction value (ppv was 94.11%. These data indicate that the recombinant protein GRA1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis.

  13. [The comparison of two newborn cytomegalovirus IgG antibody screening ELISA kits].

    Science.gov (United States)

    Zhang, Shun-Xian; He, Xiao-Zhou; Wang, Shi-Wen; Wang, Xiao-Fang

    2013-10-01

    This study compared two newborn Cytomegalovirus (CMV) IgG antibody screening ELISA kits and evaluated the detection effectiveness of Abnova kit. CMV IgG antibodies were detected by both SeraQuest and Abnova kits from dried blood spot (DBS) samples of 488 newborn heel sticks. The detection abilities of these two kits were compared in different sample dilution concentrations. Relative detection effectiveness of the Abnova kit was defined by statistical method using the SeraQuest kit as a point of comparison. Compared to the SeraQuest screening test kit, the Abnova kit revealed a sensitivity of 98.9%, specificity of 78.6%, positive predictive value of 99.3%, negative predictive value of 68.8%, and the coincidence rate for these two screening test kits at 98.3%. The consistency check of both kits based on interpretation of the kappa statistic was relatively good. For the Abnova kit, the "area under the ROC curve" was 0.887, which indicates moderate accuracy. Abnova kit can be applied to newborn screening for congenital CMV infections. However, repeating the test for ambiguous results is suggested to increase the specificity and negative predictive value.

  14. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    Science.gov (United States)

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgGantibodies. PMID:23567210

  15. Identifying anti-Toxocara IgG antibodies in horses of Mexico

    Directory of Open Access Journals (Sweden)

    R. Heredia

    Full Text Available ABSTRACT Both the presence of owned dogs and stray dogs allows the spread of Toxocara, a parasite whose eggs can be found in soil, water and food. Animals, including horses, serve as definitive and paratenic hosts. In México, where consumption of horse meat is common, Toxocara is a zoonotic parasite. The aim of this study was to identify the presence of anti-Toxocara antibodies in work horses and horses intended for human consumption by ELISA. ELISA was chosen for analysis as paratenic hosts do not shed Toxocara eggs in their feces. Blood samples were collected from a total of 188 horses, 94 of which were work horses and 94 horses from the slaughter house. Samples were analyzed by ELISA, and the general equine seroprevalence was found to be 44.6% (n = 188. Adult horses for slaughter had a 61.7% greater presence of anti-Toxocara antibodies (p = 0.006. Toxocara IgG antibodies were found in horses, confirming that horses are paratenic hosts and possible sources of infection for other animals and people.

  16. A fluorescence sedimentation assay for dsDNA antibodies

    DEFF Research Database (Denmark)

    Duus, K; Draborg, A H; Güven, E

    2017-01-01

    The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other...... on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

  17. Characterization of antibody response in neuroinvasive infection caused by Toscana virus.

    Science.gov (United States)

    Pierro, A; Ficarelli, S; Ayhan, N; Morini, S; Raumer, L; Bartoletti, M; Mastroianni, A; Prati, F; Schivazappa, S; Cenni, P; Vocale, C; Rossini, G; Gaibani, P; Sambri, V; Landini, M P; Lewis, R E; Charrel, R N; Varani, S

    2017-11-01

    Among sandfly-borne pathogens, Toscana virus (TOSV) is a prominent cause of summer meningitis in Mediterranean Europe. Here, we assessed the kinetics of anti-TOSV antibodies over time in 41 patients diagnosed with TOSV meningitis or meningoencephalitis in northeastern Italy. Acute and follow-up serum samples were collected up to 20 months after diagnosis of TOSV infection and tested for the presence of specific antibody using immunoenzymatic and indirect immunofluorescence assays. In addition, maturation of anti-TOSV IgG over time was evaluated as well as production of neutralizing antibodies. Specific IgM and IgG response was present at diagnosis in 100% of patients; TOSV-specific IgM and IgG were detected in patients' sera up to 6 and 20 months after diagnosis, respectively. The avidity index (AI) increased over the first month after infection in 100% of patients and most cases exceeded 60% by Day 30 post infection. The AI subsequently plateaued then declined at 20 months after diagnosis. Finally, neutralization assay to TOSV was performed in 217 sera collected from 41 patients; 69.6% of tested samples resulted in reactive and moderate levels of neutralizing antibodies observed during all phases of infection despite high titres of total anti-TOSV IgG. Specific antibody response develops rapidly and is long-lasting for neuroinvasive TOSV infection. Serodiagnosis of neuroinvasive TOSV requires simultaneous detection of specific IgM and IgG. Moderate levels of neutralizing antibodies were maintained over the study period, while the protective role of antibodies lacking neutralizing activity is unclear and requires further evaluation. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  18. Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection.

    Science.gov (United States)

    Wood, Britta A; Carver, Scott; Troyer, Ryan M; Elder, John H; VandeWoude, Sue

    2013-10-31

    Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases. © 2013.

  19. High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Koch, Claus; Jensen, Charlotte H

    2004-01-01

    were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging...... of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54......) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were...

  20. Study on quantification of HBs-antibody by immunoradiometric assay

    International Nuclear Information System (INIS)

    Kondo, Yuichi; Itoi, Yoshihiro; Kajiyama, Shizuo

    1989-01-01

    Quantification of HBs-antibody assay was carried out using a commercialized assay kit and standard solutions of HBs-antibody recognised as 1 st reference preparation of hepatitis B immunogloblin by WHO. Standard curve of HBs-antibody was drawn with the function of 3D-spline and the correlation factor was obtained as r = 0.999. Coefficient of intra-assay variance was 3.8 % and that of inter-assay variance was 7.8 %. Dilution tests showed satisfactory results in the range of 2-16 times. Correlation between value of cut-off indices and concentration of HBs-antibody was obtained as the formula of y = 2.599 x-3.894 (r = 0.992) and 2.1 of cut-off index corresponded to about 5 mIU/ml of HBs-antibody concentration. (author)

  1. IgG2 antibodies against a clinical grade Plasmodium falciparum CSP vaccine antigen associate with protection against transgenic sporozoite challenge in mice.

    Directory of Open Access Journals (Sweden)

    Robert Schwenk

    Full Text Available The availability of a highly purified and well characterized circumsporozoite protein (CSP is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP. A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE of CS/D in combination with the Toll-Like Receptor 4 (TLR4 agonist Glucopyranosyl Lipid A (GLA/SE, or one of two TLR7/8 agonists: R848 (un-conjugated or 3M-051 (covalently conjugated. Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive T(H1/T(H2 response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants

  2. Homogeneous immunoassay for human IgG using oriented hen egg IgY immobilized on gold sol nanoparticles

    International Nuclear Information System (INIS)

    Yeritsyan, H.E.; Gasparyan, V.K.

    2012-01-01

    Homogeneous immunoassays using (red) gold nanoparticles represent an attractive detection scheme because of the option of photometric readout. We have applied oriented immobilization of hen egg immunoglobulin Y (IgY) on gold nanoparticles when developing a homogeneous immunoassay for human IgG. In oriented immobilization, as opposed to random immobilization, the antigen binding capabilities of the antibodies are retained. It is shown that such immunoassay has significantly better sensitivity in comparison with methods based on conventional immobilization of affinity-purified antibodies. It is also shown that hen egg IgY is better suited than rabbit antibodies, because much more antibody can be immobilized on gold nanoparticles without any destabilization, probably because of the more acidic nature of these antibodies. In addition, hen egg IgY can be supplied in higher quantity and can be prepared more easily than IgG from rabbits. Bleeding and slaughtering of animals is not needed. The assay presented here has a wide detection range (30-500 ng. mL -1 ) and a limit of detection as low as 30 ng. mL -1 of human IgG. (author)

  3. Presence of Mycoplasma fermentans in the bloodstream of Mexican patients with rheumatoid arthritis and IgM and IgG antibodies against whole microorganism

    Directory of Open Access Journals (Sweden)

    Salinas Salvador

    2009-08-01

    Full Text Available Abstract Background Increasing evidence incriminates bacteria, especially Mycoplasma fermentans, as possible arthritogenic agents in humans. The purpose of this study was to investigate M. fermentans in the bloodstream of patients with rheumatoid arthritis. Methods Two hundred and nineteen blood samples from patients with rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, and healthy individuals were screened by bacterial culture and direct PCR in order to detect mycoplasmas; IgM and IgG against M. fermentans PG18 were also detected by ELISA and Immunoblotting assays in patients with rheumatoid arthritis and healthy individuals. Results Blood samples from patients with antiphospholipid syndrome and healthy individuals were negative for mycoplasma by culture or direct PCR. In blood samples from patients with systemic lupus erythematosus were detected by direct PCR M. fermentans in 2/50 (2%, M. hominis in 2/50 (2% and U. urealyticum in 1/50 (0.5%. In patients with RA M. fermentans was detected by culture in 13/87 blood samples and in 13/87 by direct PCR, however, there was only concordance between culture and direct PCR in six samples, so M. fermentans was detected in 20/87(23% of the blood samples from patients with RA by either culture or PCR. Antibody-specific ELISA assay to M. fermentans PG18 was done, IgM was detected in sera from 40/87 patients with RA and in sera of 7/67 control individuals, IgG was detected in sera from 48/87 RA patients and in sera from 7/67 healthy individuals. Antibody-specific immunoblotting to M. fermentans PG18 showed IgM in sera from 35/87 patients with RA and in sera from 4/67 healthy individuals, IgG was detected in sera from 34/87 patients and in sera from 5/67 healthy individuals. Conclusion Our findings show that only M. fermentans produce bacteremia in a high percentage of patients with RA. This finding is similar to those reported in the literature. IgM and IgG against M

  4. Characterization of isotypes of antibody response against leishmania parasite

    International Nuclear Information System (INIS)

    Elassad, Asma M.S.; Ghalib, Hashim W.; Younis, Saddia A.

    1994-01-01

    In this study an enzyme linked immunosorbent assay (ELIZA) was developed to detect IgG,IgM and IgA response in visceral leishmaniasis patients (VL) against L.donovain and L. major antigens compared to control groups; cutaneous leishmaniasis patients (CL), mucosal leishmaniasis patients (ML), patients with other tropical diseases and healthy controls.Highly specific IgG were found in VL patients with test specificity (93.7%) and sensitivity(93.4%). A moderate IgG were found in VL patients but non-specific while no IgA were detected in all studied groups. Also VL patients showed high specificity and sensitivity (95.2 and 96.6% respectively) against L.major antigen.The distribution of IgG subclasses (IgG1,IgG2,IgG3 and IgG4) antibodies in VL patients were assayed.IgG3 showed the highest specificity and sensitivity and titers followed by IgG1.Also the diagnostic value of ELIZA test for different leishmaniasis forms were discussed. (Author)

  5. Performance evaluation of a novel chemiluminescence assay for detection of anti-GBM antibodies: an international multicenter study.

    Science.gov (United States)

    Mahler, Michael; Radice, Antonella; Sinico, Renato A; Damoiseaux, Jan; Seaman, Andrea; Buckmelter, Kristen; Vizjak, Alenka; Buchner, Carol; Binder, Walter L; Fritzler, Marvin J; Cui, Zhao

    2012-01-01

    Autoantibodies to the non-collagen region (NC1) of the alpha-3 subunit of collagen IV represent a serological hallmark in the diagnosis of Goodpasture's syndrome (GPS). The objective of our study was to carefully analyze the performance characteristics of a novel anti-glomerular basement membrane (GBM) chemiluminescence immunoassay (CIA). Sera from patients with GPS (n = 90) were collected from four clinical centers. Samples from different disease groups (n = 397) and healthy individuals (n = 400) were used as controls. All samples were tested for anti-GBM antibodies by a rapid, random access CIA (QUANTA Flash™ GBM). Most of the samples were also tested using other methods including different commercial anti-GBM IgG assays and research assays for anti-GBM IgA and IgM. The sensitivity and specificity of the novel CIA was 95.6% [95% confidence interval (CI) 89.0-98.8%] and 99.6% (95% CI 98.9-99.9%), respectively. Receiver operating characteristic analysis showed good discrimination between GPS patients and controls. The area under the curve was 0.98 (CI 0.96-1.0). The three anti-GBM antibody-positive samples from the control group were from two healthy individuals and one human immunodeficiency virus (HIV)-infected patient. All three individuals had low levels of anti-GBM antibodies [20, 24 and 25 chemiluminescent unit (CU), cutoff 20 CU]. When the results of the new CIA were compared to other methods, good agreement was observed: 95.8% (kappa = 0.92) versus EliA™ GBM, 97.4% (kappa = 0.95) versus both BINDAZYME™ Anti-GBM and QUANTA Lite® GBM. Anti-GBM IgA was detectable in low concentrations in patients with GPS and was associated with anti-GBM IgG but was less useful in discriminating GPS patients and controls. No discrimination was found for anti-GBM IgM. The novel QUANTA Flash™ GBM CIA demonstrated good sensitivity and specificity and had good agreement with other methods. Our data confirm that ∼5% of patients with GPS do not have detectable levels of

  6. Possible role of specific immunoglobulin M antibodies to Plasmodium falciparum antigens in immunoprotection of humans living in a hyperendemic area, Burkina Faso

    DEFF Research Database (Denmark)

    Boudin, C; Chumpitazi, B; Dziegiel, M

    1993-01-01

    of antibodies to crude extracts of Plasmodium falciparum (IgG or IgM antisomatic and IgG antiexoantigens) were tested by IFI or enzyme-linked immunosorbent assay and were followed up according to the fluctuations of the parasite densities. Specific IgG antibodies had the same evolution as parasite densities....... Group 3 was composed of immunoprotected adults. Specific IgM and IgG antibodies to crude extracts or a recombinant antigen (glutamate-rich protein) of P. falciparum were tested. Specific IgM antibodies were lower in group 1 (nonimmune) than in groups 2 (semiimmune) and 3 (immunoprotected). Furthermore...

  7. The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production.

    Science.gov (United States)

    Ushijima, Miho; Uruno, Takehito; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Kamikaseda, Yasuhisa; Kunimura, Kazufumi; Sakata, Daiji; Okada, Takaharu; Fukui, Yoshinori

    2018-01-01

    A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab') 2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.

  8. Quantitation of parasite-specific human IgG and IgE in sera: evaluation of solid-phase RIA and ELISA methodology

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R G [Johns Hopkins Univ., Baltimore, MD (USA). Dept. of Medicine; Hussain, R; Ottesen, E A [National Inst. of Allergy and Infectious Diseases, Bethesda, MD (USA); Adkinson, Jr, N F [Johns Hopkins Univ., Baltimore, MD (USA). School of Medicine

    1981-07-17

    The authors have developed a non-competitive solid-phase radioimmunoassay (SPRIA) to quantitate both human IgE and IgG antibodies against soluble adult antigens of Brugia malayi (B.m.), a filarial parasite causing extensive infection throughout the tropics. Previously enzyme-linked immunosorbent assays (ELISA) had been used to detect ..mu..g/ml levels of IgG anti-B.m., but IgE antibodies were difficult to detect in this system. Since the SPRIA successfully quantitates both IgG and IgE anti-B.m., they sought to examine the reasons for the SPRIA's apparent superiority in detecting IgE anti-B.m. by extracting specific IgG from sera with high levels of IgE and IgG anti-B.m. antibodies. IgE anti-B.m. was then quantitated in these sera using both the SPRIA and ELISA methods. Results indicate that IgG anti-B.m. does not interfere with detection of specific IgE antibody in the SPRIA but does interfere in the ELISA. While ELISA permits detection of IgE anti-B.m. in the absence of competing IgG anti-B.m., as levels of specific IgG increase, the IgE is no longer detectable. These differences between SPRIA and ELISA can be explained by the SPRIA's antigen excess conditions which assure that there are sufficient antigens both to detect all anti-B.m. antibodies present in the serum and to adequately represent all antigen specificities in the crude B.m. extract. Their findings commend the use of SPRIA methods over ELISA in assessment of B.m.-specific IgE antibody in filariasis and indicate a potential role for SPRIA methods in absolute quantitation of specific serum antibodies.

  9. Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

    Science.gov (United States)

    Howie, Heather L; Delaney, Meghan; Wang, Xiaohong; Er, Lay See; Vidarsson, Gestur; Stegmann, Tamara C; Kapp, Linda; Lebedev, Jenna N; Wu, Yanyun; AuBuchon, James P; Zimring, James C

    2016-12-01

    Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported. © 2016 AABB.

  10. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

    Directory of Open Access Journals (Sweden)

    SANT'ANNA Anadayr L.M.

    2002-01-01

    Full Text Available The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE, Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140 have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05 was seen between IgG antibody prevalence in male (27.8% and female (35.5% patients. Anti-B19 IgG antibodies were more frequent in older (37.6% than younger (28.2% than 20 years old patients, although this difference had no statistical significance (p > 0.05. Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  12. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    Science.gov (United States)

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests

  13. Liver-X-receptor activator prevents homocysteine-induced production of IgG antibodies from murine B lymphocytes via the ROS-NF-κB pathway

    International Nuclear Information System (INIS)

    Chang Lina; Zhang, Zhenmin; Li Wenjing; Dai Jing; Guan Youfei; Wang Xian

    2007-01-01

    Our previous study showed that homosysteine (Hcy) promotes proliferation of mouse splenic B lymphocytes. In this study, we investigated whether Hcy could stimulate the production of IgG antibodies. Hcy significantly increased the production of IgG antibodies from resting B lymphocytes. B lymphocytes from ApoE-knockout mice with hyperhomocysteinemia showed elevated IgG secretion at either the basal Hcy level or in response to lipopolysaccharide. Hcy promoted reactive oxygen species (ROS) formation, and free radical scavengers, MnTMPyP decreased Hcy-induced IgG secretion. The inhibitor of NF-κB (MG132) also significantly reduced Hcy-induced IgG secretion. Furthermore, Hcy-induced formation of ROS, activation of NF-κB, and secretion of IgG could be inhibited by the liver-X-receptor (LXR) agonist TO 901317. Thus, our data provide strong evidence that HHcy induces IgG production from murine splenic B lymphocytes both in vitro and in vivo. The mechanism might be through the ROS-NF-κB pathway and can be attenuated by the activation of LXR

  14. Naturally acquired antibodies target the glutamate-rich protein on intact merozoites and predict protection against febrile malaria

    DEFF Research Database (Denmark)

    Kana, Ikhlaq Hussain; Adu, Bright; Tiendrebeogo, Régis Wendpayangde

    2017-01-01

    febrile malaria. Similarly, GLURP-specific antibodies previously shown to be protective against febrile malaria in this same cohort were significantly associated with OP activity in this study. GLURP-specific antibodies recognized merozoites and also mediated OP activity. Conclusions.: These findings......Background.: Plasmodium species antigens accessible at the time of merozoite release are likely targets of biologically functional antibodies. Methods.: Immunoglobulin G (IgG) antibodies against intact merozoites were quantified in the plasma of Ghanaian children from a longitudinal cohort using...... a novel flow cytometry-based immunofluorescence assay. Functionality of these antibodies, as well as glutamate-rich protein (GLURP)-specific affinity-purified IgG from malaria hyperimmune Liberian adults, was assessed by the opsonic phagocytosis (OP) assay. Results.: Opsonic phagocytosis activity...

  15. Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis

    DEFF Research Database (Denmark)

    Hjelholt, Astrid; Carlsen, Thomas Gelsing; Deleuran, Bent Winding

    2013-01-01

    Introduction: Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. SpA is suggested triggered by bacterial infection, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since...... against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60...... was seen, and there was no indication of cross-reaction. Conclusion: These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported...

  16. IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

    Science.gov (United States)

    Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

    2009-10-01

    Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.

  17. AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

    Science.gov (United States)

    Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

    2017-09-29

    Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

  18. Enzyme-linked immunosorbent Assay for detecting of antibody to canine distemper virus

    Directory of Open Access Journals (Sweden)

    Sudarisman

    2006-03-01

    Full Text Available Serum neutralisation test (SNT has been established for evaluating canine distemper vaccination, but until now SNT was rarely used due to the need for continuous tissue culture facilities and requires 3 days to perform. For detecting antibody to canine distemper virus, an enzyme-linked immunosorbent assay (ELISA is relatively simple and rapid seroassay. ELISA for canine immunoglobulin (Ig G antibodies to canine distemper virus (CDV was developed by using Onderstepoort strain of canine distemper virus as coating antigen. Rabbit anti canine IgG labelled with horse radish peroxidase was used as the conjugate, while phenylenediamine dihydrochloride (OPD was used as the substrate. The ELISA results were then compared with the results of the SNT, using the sera of 312 random-source dogs from West Java. The two test-results had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1 : 100 dilutions, there was a 95.5% agreement between the ELISA and SNT. Their sensitivity and spesificity were 83.9 and 98.4%. Titrated SNT and ELISA also were performed on sera from 7 dogs whose lifetime medical histories were known. The antibodies were inclining up after two months of post vaccination, where the titre was not in zero/lower position at the day of vaccination. However, antibody zero or low position were found at 28 days post vaccination. All of the results indicated that ELISA can be used for evaluating antibody to canine distemper virus response, replacing the SNT.

  19. Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients.

    Science.gov (United States)

    Parra-Sánchez, Manuel; Zakariya-Yousef Breval, Ismail; Castro Méndez, Carmen; García-Rey, Silvia; Loza Vazquez, Ana; Úbeda Iglesias, Alejandro; Macías Guerrero, Desiree; Romero Mejías, Ana; León Gil, Cristobal; Martín-Mazuelos, Estrella

    2017-08-01

    Testing for Candida albicans germ-tube antibody IFA IgG assay (CAGTA) is used to detect invasive candidiasis infection. However, most suitable assays lack automation and rapid single-sample testing. The CAGTA assay was adapted in an automatic monotest system (invasive candidiasis [CAGTA] VirClia ® IgG monotest (VirClia ® ), a chemiluminescence assay with ready-to-use reagents that provides a rapid objective result. CAGTA assay was compared with the monotest automatic VirClia ® assay in order to establish the diagnostic reliability, accuracy, and usefulness of this method. A prospective study with 361 samples from 179 non-neutropenic critically ill adults patients was conducted, including 21 patients with candidemia, 18 with intra-abdominal candidiasis, 84 with Candida spp. colonization, and 56 with culture-negative samples, as well as samples from ten healthy subjects. Overall agreement between the two assays (CAGTA and VirCLIA) was 85.3%. These assays were compared with the gold-standard method to determine the sensitivity, specificity as well as positive and negative predictive values. In patients with candidemia, values for CAGTA and VirCLIA assays were 76.2 versus 85.7%, 80.3 versus 75.8%, 55.2 versus 52.9%, and 91.4 versus 94.3%, respectively. The corresponding values in patients with intra-abdominal candidiasis were 61.1 versus 66.7%, 80.3 versus 75.8%, 45.8 versus 42.9%, and 88.3 versus 89.3%, respectively. No differences were found according to the species of Candida isolated in culture, except for Candida albicans and C. parapsilosis, for which VirClia ® was better than CAGTA. According to these results, the automated VirClia ® assay was a reliable, rapid, and very easy to perform technique as tool for the diagnosis invasive candidiasis.

  20. The quantitation of parasite-specific human IgG and IgE in sera: evaluation of solid-phase RIA and ELISA methodology

    International Nuclear Information System (INIS)

    Hamilton, R.G.; Adkinson, N.F. Jr.

    1981-01-01

    The authors have developed a non-competitive solid-phase radioimmunoassay (SPRIA) to quantitate both human IgE and IgG antibodies against soluble adult antigens of Brugia malayi (B.m.), a filarial parasite causing extensive infection throughout the tropics. Previously enzyme-linked immunosorbent assays (ELISA) had been used to detect μg/ml levels of IgG anti-B.m., but IgE antibodies were difficult to detect in this system. Since the SPRIA successfully quantitates both IgG and IgE anti-B.m., they sought to examine the reasons for the SPRIA's apparent superiority in detecting IgE anti-B.m. by extracting specific IgG from sera with high levels of IgE and IgG anti-B.m. antibodies. IgE anti-B.m. was then quantitated in these sera using both the SPRIA and ELISA methods. Results indicate that IgG anti-B.m. does not interfere with detection of specific IgE antibody in the SPRIA but does interfere in the ELISA. While ELISA permits detection of IgE anti-B.m. in the absence of competing IgG anti-B.m., as levels of specific IgG increase, the IgE is no longer detectable. These differences between SPRIA and ELISA can be explained by the SPRIA's antigen excess conditions which assure that there are sufficient antigens both to detect all anti-B.m. antibodies present in the serum and to adequately represent all antigen specificities in the crude B.m. extract. Their findings commend the use of SPRIA methods over ELISA in assessment of B.m.-specific IgE antibody in filariasis and indicate a potential role for SPRIA methods in absolute quantitation of specific serum antibodies. (Auth.)

  1. A polyethylene glycol radioimmunoprecipitation assay for human immunoglobulin G

    International Nuclear Information System (INIS)

    Waller, S.J.; Taylor, R.P.; Andrews, B.S.

    1979-01-01

    A polyethylene glycol (PEG) radioimmunoprecipitation assay for human IgG is described that is sufficiently sensitive to detect 0.5 ng of IgG. This model antibody-antigen system was also used to study the stoichiometries of PEG-precipitation complexes. The results suggest that the presence of PEG may affect the stoichiometry of the complexes which precipitate from solution. (Auth.)

  2. Assessment of pathogenesis of infective endocarditis by plasma IgG antibody titer test against periodontal bacteria.

    Science.gov (United States)

    Isoshima, Daichi; Yamashiro, Keisuke; Matsunaga, Kazuyuki; Shinobe, Michitaka; Nakanishi, Nagako; Nakanishi, Izumi; Omori, Kazuhiro; Yamamoto, Tadashi; Takashiba, Shogo

    2017-10-01

    Oral bacteria cause infective endocarditis (IE), so severe periodontitis is thought to be high risk for IE. We suggest the identification of high-risk patients by an IgG antibody titer test against periodontal bacteria might become common screening test.

  3. Potential of Murine IgG1 and Human IgG4 to Inhibit the Classical Complement and Fcγ Receptor Activation Pathways

    Directory of Open Access Journals (Sweden)

    Gina-Maria Lilienthal

    2018-05-01

    Full Text Available IgG antibodies (Abs mediate their effector functions through the interaction with Fcγ receptors (FcγRs and the complement factors. The main IgG-mediated complement activation pathway is induced through the binding of complement C1q to IgG Abs. This interaction is dependent on antigen-dependent hexamer formation of human IgG1 and IgG3 to increase the affinity for the six-headed C1q molecule. By contrast, human IgG4 fails to bind to C1q. Instead, it has been suggested that human IgG4 can block IgG1 and IgG3 hexamerization required for their binding to C1q and activating the complement. Here, we show that murine IgG1, which functionally resembles human IgG4 by not interacting with C1q, inhibits the binding of IgG2a, IgG2b, and IgG3 to C1q in vitro, and suppresses IgG2a-mediated complement activation in a hemolytic assay in an antigen-dependent and IgG subclass-specific manner. From this perspective, we discuss the potential of murine IgG1 and human IgG4 to block the complement activation as well as suppressive effects of sialylated IgG subclass Abs on FcγR-mediated immune cell activation. Accumulating evidence suggests that both mechanisms seem to be responsible for preventing uncontrolled IgG (autoAb-induced inflammation in mice and humans. Distinct IgG subclass distributions and functionally opposite IgG Fc glycosylation patterns might explain different outcomes of IgG-mediated immune responses and provide new therapeutic options through the induction, enrichment, or application of antigen-specific sialylated human IgG4 to prevent complement and FcγR activation as well.

  4. Comparison of IgG and IgA Antibodies Titrations against Helicobacter Pylori in Urban and Rural Populations in Mazandaran Province

    Directory of Open Access Journals (Sweden)

    Mina Owrang

    2014-06-01

    Full Text Available Background & objective: The infection caused by Helicobacter pylori (H. pylori is one of the most common bacterial gastrointestinal diseases throughout the world. Based on the role of H. pylori in a variety of diseases such as gastrointestinal and lymphoma, present study is aimed to consider the concentration of IgA and IgG against H. pylori in both rural and urban populations and then its relationship with some demographic characteristics. Materials & Methods: In this descriptive study, 400 sera samples were collected from both genders at the Sari treatment- health center. After blood collection, the concentration of IgG and IgA against H. pylori was measured by ELISA kit. Results: There was no significant difference in antibodies titration between men and women. Approximately 18.5% of males and 16.5% of females were positive regarding to IgA and 70.2% of men and 66.7% of women were positive regarding to IgG. The mean of antibodies in rural populations (0.87±0.35 was significantly (p<0.001 higher than those in urban populations (0.78±0.41. The mean of antibodies in patients who had a history of gastrointestinal infection was significantly higher than others (p<0.05. Conclusion: Due to the high level of IgA and IgG antibodies in studied populations, especially in rural people, and lack of symptoms in patients, the screen of positive serologic populations can be helpful for the management and control of infections caused by H. pylori.

  5. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    Science.gov (United States)

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  6. IgG subclass reactivity to Trypanosoma cruzi in chronic chagasic patients.

    Science.gov (United States)

    Hernández-Becerril, N; Nava, A; Reyes, P A; Monteón, V M

    2001-01-01

    The anti-Trypanosoma cruzi antibodies isotype profile in Chagas' disease has been studied in relation to different clinical manifestations. A high titer of IgG anti-T. cruzi antibodies is found in patients with cardiac involvement, while a high titer of IgA anti-T. cruzi antibodies is associated with digestive forms. The aim of this work was to analyze the IgG subclass reactivity of anti-T. cruzi antibodies in patients with chronic Chagasic cardiomyopathy. Twelve consecutive chagasic patients were analyzed for IgG subclass reactivity to a T. cruzi antigenic extract. They had a complete clinical evaluation, peripheral EKG, echocardiography, left ventriculogram, and coronariography. All patients came from rural areas of Mexico and had lived in endemic zones for over seven years. They presented left ventricular endsystolic dimension above 42 mm in 58% (7/12) and ejection fraction below 50% in 58% (7/12). We found that IgG1 and IgG2 anti-T. cruzi antibodies showed higher titer than IgG3 antibodies, with consistently low titer of IgG4 antibodies. Expression of the four IgG subclasses of anti-T. cruzi antibodies suggest a mixed Th1/Th2-like immune response under a probably continuous chronic antigenic stimulation. On the other hand, high levels of IgG2 anti-T. cruzi antibodies showed a tendency to be associated with severe cardiomegaly. Our results suggest that a mixed Th1/Th2-like immune response may take place in chronic chagasic patients under a chronic antigenic stimulation.

  7. The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production

    Directory of Open Access Journals (Sweden)

    Miho Ushijima

    2018-02-01

    Full Text Available A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR, which triggers activation of B cells and differentiation into plasma cells (PCs. Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab′2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2–Rac axis in PC differentiation and IgG antibody responses.

  8. Prohibitin Is Involved in Patients with IgG4 Related Disease.

    Directory of Open Access Journals (Sweden)

    Hongwu Du

    Full Text Available IgG4-related disease (IgG4-RD is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD.Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF mass spectrometry. After the cloning and expression of recombinant putative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of confirmed clinical samples.One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%, Mikulicz's disease (8/15; 53.3%, retroperitoneal fibrosis (6/11; 54.5%, other probable IgG4-RD (26/29; 89.7% and Sjögren's syndrome (4/30; 13.3% but not in apparently healthy donors (1/70; 1.4%.An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pancreatitis, Mikulicz's disease and retroperitoneal fibrosis.

  9. Immune thrombocytopenia. Use of a Coombs antiglobulin test to detect IgG and C3 on platelets

    International Nuclear Information System (INIS)

    Cines, D.B.; Schreiber, A.D.

    1979-01-01

    We applied a radiolabeled Coombs antiglobulin test to the diagnosis and management of immune thrombocytopenia in adults and children. This assay substantiated that the majority of patients with idiopathic thrombocytopenic purpura have increased levels of IgG on their platelets. Platelets from a patient with the post-transfusion-purpura syndrome also carried increased IgG, indicating a role for IgG antibody or IgG-containing immune complexes in the destruction of host platelets in this disease. The radiolabeled Coombs test provides a general means to help diagnose, manage, and study immune platelet disorders

  10. The usefulness of casein-specific IgE and IgG4 antibodies in cow's milk allergic children

    Directory of Open Access Journals (Sweden)

    Ito Komei

    2012-01-01

    Full Text Available Abstract Background Cow's milk allergy is one of the most common food allergies among younger children. We investigated IgE antibodies to milk, and IgE and IgG4 antibodies to casein, α-lactalbumin and β-lactoglobulin in cow's milk allergic (CMA and non-allergic (non-CMA children in order to study their clinical usefulness. Methods Eighty-three children with suspected milk allergy (median age: 3.5 years, range: 0.8-15.8 years were diagnosed as CMA (n = 61 or non-CMA (n = 22 based on an open milk challenge or convincing clinical history. Their serum concentrations of allergen-specific (s IgE and IgG4 antibodies were measured using ImmunoCAP®. For the sIgG4 analysis, 28 atopic and 31 non-atopic control children were additionally included (all non-milk sensitized. Results The CMA group had significantly higher levels of milk-, casein- and β-lactoglobulin-sIgE antibodies as compared to the non-CMA group. The casein test showed the best discriminating performance with a clinical decision point of 6.6 kUA/L corresponding to 100% specificity. All but one of the CMA children aged > 5 years had casein-sIgE levels > 6.6 kUA/L. The non-CMA group had significantly higher sIgG4 levels against all three milk allergens compared to the CMA group. This was most pronounced for casein-sIgG4 in non-CMA children without history of previous milk allergy. These children had significantly higher casein-sIgG4 levels compared to any other group, including the non-milk sensitized control children. Conclusions High levels of casein-sIgE antibodies are strongly associated with milk allergy in children and might be associated with prolonged allergy. Elevated casein-sIgG4 levels in milk-sensitized individuals on normal diet indicate a modified Th2 response. However, the protective role of IgG4 antibodies in milk allergy is unclear.

  11. On the role of IgG4 in inflammatory conditions: lessons for IgG4-related disease

    NARCIS (Netherlands)

    Trampert, David C.; Hubers, Lowiek M.; van de Graaf, Stan F. J.; Beuers, Ulrich

    2017-01-01

    The pathophysiology of immunoglobulin G4-related disease (IgG4-RD) and its most common manifestations, IgG4-associated (sclerosing) cholangitis and autoimmune pancreatitis, remains largely unknown, but IgG4 is presumably involved. IgG4 is a promiscuous antibody, which could be directly pathogenic,

  12. Measurement of anti-IgA antibodies by a two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Homburger, H.A.; Smith, J.R.; Jacob, G.L.; Laschinger, C.; Naylor, D.H.; Pineda, A.A.

    1981-01-01

    To enable the detection of IgG class, anti-IgA antibodies and to investigate the possible occurrence of IgE class, anti-IgA antibodies, we developed a solid phase immunoradiometric assay, which uses purified IgA coupled covalently to microcrystalline cellulose as an immunosorbent. Radiolabeled, Fc specific anti-IgG and anti-IgE antibodies were used to detect specific aIgA after incubation of test sera or controls with the immunosorbent. IgG-aIgA were detected by the IRA in 100 and 67% of control sera with class specific and limited specificity aIgA. The IRA was sensitive to approximately two ng of class specific IgG-aIgA. IgG-aIgA also were detected by IRA in 7.9% of sera from patients with urticarial transfusion reactions and 73% of sera from patients with ataxia telangiectasia and IgA deficiency. Sera from 50 normal blood donors did not have detectable IgG-aIgA. Tests for IgE-aIgA were negative in all cases, including control sera with class specific IgG-aIgA. We conclude that the IRA is a sensitive and reproducible method for detection of class specific and limited specificity IgG-aIgA, and that IgE-aIgA do not mediate urticarial transfusion reactions

  13. Evaluation of anti-Schistosoma mansoni igG antibodies in patients with chronic schistosomiasis mansoni before and after specific treatment Avaliação da presença de anticorpos IgG anti-Schistosoma mansoni no soro de pacientes com esquistossomose mansônica crônica, antes e após tratamento específico

    Directory of Open Access Journals (Sweden)

    Célia Maria V. VENDRAME

    2001-06-01

    Full Text Available The circumoval precipitin test (COPT, enzyme-linked immunosorbent assay (ELISA and the immunoblotting anti-adult worm antigen (AWA and soluble egg antigen (SEA tests were applied to 17 chronically schistosome-infected patients for the detection of anti-Schistosoma mansoni antibodies before and on four occasions after oxamniquine administration over a period of six months. Compared to a control group, schistosomiasis patients showed high levels of IgG antibodies in AWA and SEA-ELISA. A decrease in IgG levels was observed six months after treatment, although negative reactions were not obtained. Significant decreases in IgG1, IgG3 and, mainly, IgG4, but not anti-SEA IgG2 levels were observed six months after treatment, again without negativity. Analysis of anti-AWA IgG antibodies by immunoblotting before treatment showed a 31 kDa strand in 14 patients (82% which disappeared in three cases up to six months after treatment; furthermore, anti-SEA IgG antibodies showed the same band in nine patients (53% before treatment, which disappeared in only four cases up to six months after treatment.Em 17 pacientes com infecção crônica por Schistosoma mansoni utilizaram-se os testes de reação periovular, imunoenzimático (ELISA e imunoblotting, empregando-se antígenos obtidos a partir de vermes adultos (AWA ou de ovos de S. mansoni (SEA, para detecção de anticorpos anti-S. mansoni, antes e em quatro ocasiões após tratamento com oxamniquine. Quando cotejados a grupo controle os pacientes esquistossomóticos revelaram altos níveis séricos de anticorpos IgG nos testes ELISA (anti-AWA e anti-SEA, não se observando, porém, negativação até seis meses após tratamento específico. Encontrou-se, entretanto, decréscimo significativo, sem negativação, dos níveis de IgG1, IgG3 e, principalmente, IgG4, quando se utilizou antígeno solúvel obtido a partir de ovos de S. mansoni (SEA, seis meses após administração de oxamniquine. O mesmo não foi

  14. Characterization of isotypes of antibody response against leishmania parasite

    Energy Technology Data Exchange (ETDEWEB)

    Elassad, Asma M.S.; Ghalib, Hashim W [Medical Parasitology Project NIH/Sudan, Khartoum (Sudan); Younis, Saddia A [Department of Zoology, Faculty of Science, University of Khartoum, Khartoum (Sudan)

    1994-12-01

    In this study an enzyme linked immunosorbent assay (ELIZA) was developed to detect IgG,IgM and IgA response in visceral leishmaniasis patients (VL) against L.donovain and L. major antigens compared to control groups; cutaneous leishmaniasis patients (CL), mucosal leishmaniasis patients (ML), patients with other tropical diseases and healthy controls.Highly specific IgG were found in VL patients with test specificity (93.7%) and sensitivity(93.4%). A moderate IgG were found in VL patients but non-specific while no IgA were detected in all studied groups. Also VL patients showed high specificity and sensitivity (95.2 and 96.6% respectively) against L.major antigen.The distribution of IgG subclasses (IgG1,IgG2,IgG3 and IgG4) antibodies in VL patients were assayed.IgG3 showed the highest specificity and sensitivity and titers followed by IgG1.Also the diagnostic value of ELIZA test for different leishmaniasis forms were discussed. (Author). 18 refs., 1 fig., 3 tabs.

  15. Isolation and characterization of a monoclonal anti CK-2 alpha subunit antibody of the IgG1 subclass

    DEFF Research Database (Denmark)

    Schmidt-Spaniol, I; Boldyreff, B; Issinger, O G

    1992-01-01

    A monoclonal antibody was produced against the recombinant human alpha subunit of CK-2. The antibody was of the IgG1 subclass and it was isolated from serum-free cell culture media and purified by affinity chromatography on Protein G Sepharose. The antibody can be used to detect specifically the CK......-2 alpha subunit in immunoblots from tissue extracts. An ELISA detection test was also established which also allows the identification of the CK-2 alpha subunit....

  16. An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

    Directory of Open Access Journals (Sweden)

    Hempel Franziska

    2012-09-01

    Full Text Available Abstract Background Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. Results In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. Conclusions Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.

  17. Performance evaluation of four type-specific commercial assays for detection of herpes simplex virus type 1 antibodies in a Middle East and North Africa population.

    Science.gov (United States)

    Aldisi, Rana S; Elsidiq, Malaz S; Dargham, Soha R; Sahara, Afifah S; Al-Absi, Enas S; Nofal, Mariam Y; Mohammed, Layla I; Abu-Raddad, Laith J; Nasrallah, Gheyath K

    2018-03-22

    The number of diagnostic assays for the detection of herpes simplex virus type 1 (HSV-1) antibodies has increased over the years. However, their performance characteristics could vary among global populations. To investigate performance of two commercial ELISA kits, HerpeSelect ® 1 ELISA and Euroimmun Anti-HSV-1 (gC1) ELISA (IgG); and two commercial immunoblot (IB)/Western blot (WB) assays, HerpeSelect ® 1 and 2 Immunoblot IgG, and Euroimmun Anti-HSV-1/HSV-2 gG2 Euroline-WB (IgG/IgM); in detecting HSV-1 antibodies in a Middle East and North Africa (MENA) population. Blood specimens were collected from blood donors in Doha, Qatar, June 2013-2016. Twenty specimens were randomly selected from 10 MENA nationalities (Egypt, Iran, Jordan, Lebanon, Pakistan, Palestine, Qatar, Sudan, Syria, and Yemen; total = 200), and tested for HSV-1 antibodies. Across all six comparisons between assays, positive percent agreement ranged between 95.7% (95% CI: 91.4-98.3%) and 100.0% (95% CI: 97.8-100.0%). Negative percent agreement ranged between 86.2% (95% CI: 68.3-96.1%) and 96.2% (95% CI: 80.4-99.9%). Overall percent agreement ranged between 95.7% (95% CI: 91.7-97.8%) and 99.4% (95% CI: 96.7-99.9%). Cohen's kappa statistic ranged between 0.84 (95% CI: 0.73-0.95) and 0.98 (95% CI: 0.93-1.00). Compared against IB/WB, HerpeSelect ® and Euroimmun had sensitivities and specificities >96% and >86%, respectively. Positive and negative predictive values were >97% and >83%, respectively. The assays showed excellent concordance with one another, and with a high kappa statistic. The ELISA kits demonstrated robust diagnostic performance compared to the IB/WB assays. These findings support the assays' utility in clinical diagnosis and research in MENA populations. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

    International Nuclear Information System (INIS)

    Martins, Estefania M.N.; Andrade, Antero S.R.; Reis, Bernardo S.; Goes, Alfredo M.

    2007-01-01

    Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

  19. Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)]. E-mail: estefaniabio@yahoo.com.br; antero@cdtn.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia]. E-mail: brsgarbi@mono.icb.ufmg.br; goes@mono.icb.ufmg.br

    2007-07-01

    Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

  20. An enzyme-linked immunosorbent assay for the quantification of serum platelet-bindable IgG.

    Science.gov (United States)

    Howe, S E; Lynch, D M; Lynch, J M

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.

  1. New radioimmunoassay for IgM and IgG rheumatoid factors, based on a double antibody method

    Energy Technology Data Exchange (ETDEWEB)

    Nordfang, O. (Rigshospitalet, Copenhagen (Denmark)); Hoeier-Madsen, M.; Lieberkind, J. (Statens Seruminstitut, Copenhagen (Denmark)); Halberg, P. (Medical Department, Division of Rheumatology, Hvidovre Hospital, Hvidovre, Denmark)

    1981-11-30

    A new radioimmunoassay has been developed for measuring IgM and IgG rheumatoid factors. Diluted sera from donors and patients were incubated with immunoprecipitates prepared from sheep serum and rabbit anti-sheep IgG antiserum. The precipitates were washed, and radioiodinated rabbit F(ab')/sub 2/ fragments specific for human IgM or IgG were added. The precipitates were isolated by filtration and measured in a gamma counter. With this assay IgM rheumatoid factors were detected in sera from all patients with seropositive rheumatoid arthritis and in sera from 40% of patients with seronegative rheumatoid arthritis. IgG rheumatoid factors were found in sera from 68% of the seropositive and 40% of the seronegative patients. Gel filtration experiments demonstrated that it is possible to detect monomeric IgG rheumatoid factors and IgM rheumatoid factors of molecular weight smaller than pentameric IgM. Furthermore it has been shown that IgG rheumatoid factor activity is still present after reduction of IgM rheumatoid factors with dithiotreitol.

  2. Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Bødtger, Uffe; Kristensen, Peter

    2004-01-01

    Allergen-specific Fab fragments isolated from combinatorial IgE and IgG libraries are useful tools for studying allergen-antibody interactions. To characterise the interaction between different allergens and antibodies we have created recombinant human phage antibody libraries in the Fab format...

  3. The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

    Directory of Open Access Journals (Sweden)

    Mutapi Francisca

    2002-01-01

    Full Text Available The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.

  4. Fluorescent IgG fusion proteins made in E. coli

    Science.gov (United States)

    Luria, Yael; Raichlin, Dina; Benhar, Itai

    2012-01-01

    Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

  5. IgG4 antibodies in Egyptian patients with schistosomiasis

    NARCIS (Netherlands)

    Iskander, R.; Das, P. K.; Aalberse, R. C.

    1981-01-01

    Serum immunoglobulins were determined in 40 Egyptian patients with schistosomiasis. In addition to the well-established elevation in total IgE, a striking imbalance in the IgG subclass levels was found: IgG3 and IgG4 levels were markedly elevated, whereas IgG2 levels were normal. The IgG4 level did

  6. ''reverse'' solid-phase radioimmunoasssay for IgM-antibodies to hepatitis A virus

    Energy Technology Data Exchange (ETDEWEB)

    Meurman, O H; Matter, L; Krishna, R V; Krech, U H [Institute of Medical Microbiology, St. Gallen, Switzerland

    1981-01-01

    A ''reverse'' solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and /sup 125/I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sera was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar reverse immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens.

  7. Prospective estimation of IgG, IgG subclass and IgE antibodies to dietary proteins in infants with cow milk allergy. Levels of antibodies to whole milk protein, BLG and ovalbumin in relation to repeated milk challenge and clinical course of cow milk allergy

    DEFF Research Database (Denmark)

    Høst, A; Husby, S; Gjesing, B

    1992-01-01

    Prospectively, serum levels of IgE, specific IgE antibodies (AB) to whole cow milk protein (CMP), bovine se-albumin, bovine immunoglobulin, bovine lactoferrin, bovine lactalbumin and beta-lactoglobulin (BLG), IgG and IgG subclass antibodies to ovalbumin (OA) and BLG, and IgG4 RAST to CMP (bovine...... whey) were measured in 39 infants with cow milk protein allergy (CMPA) at birth (cord blood), at time of diagnosis and before and after milk challenge at the age of 12 months. Immunological measurements were also undertaken in 33 control infants without CMPA at birth, at 6 months and at 18 months...... of the type of CMPA (IgE-mediated (CMA) or non-IgE-mediated (CMI)), and irrespective of whether remission had occurred. In cord blood 25/33 (76%) of the infants with CMPA had specific IgE-AB to one or more of the bovine milk proteins indicating a prenatal intrauterine sensitization to cow milk protein. At 6...

  8. Seropositivity of Dengue Antibodies during Pregnancy

    Directory of Open Access Journals (Sweden)

    Nor Azlin Mohamed Ismail

    2014-01-01

    Full Text Available Purpose. Malaysia a dengue endemic country with dengue infections in pregnancy on the rise. The present study was aimed at determining dengue seroprevalence (IgG or IgM during pregnancy and its neonatal transmission in dengue seropositive women. Methods. Maternal with paired cord blood samples were tested for dengue antibodies (IgG and IgM using an enzyme-linked immunosorbent assay (ELISA. Maternal age, parity, occupation, ethnic group, and gestational age were recorded. Data on neonatal Apgar score and admissions to the Neonatal Intensive Care Unit (NICU were analyzed. Results. Out of 358 women recruited, about 128 (35.8% patients were seropositive. Twelve patients (3.4% had recent infections (IgM positive and another 116 women (32.4% were with past infections (IgG positive. All babies born to seropositive mothers had positive IgG paired cord blood; however, no IgM seropositivity was observed. All neonates had good Apgar scores and did not require NICU admission. Conclusion. In this study, 35.8% pregnant women were found to be dengue seropositive. However, transplacental transfer of IgG antibodies had no detrimental effect on the neonatal outcomes.

  9. Detection of Serum IgG4 Levels in Patients with IgG4-Related Disease and Other Disorders

    Science.gov (United States)

    Wang, Chenqiong; Wu, Xuefen; Miao, Ye; Xiong, Hui; Bai, Lin; Dong, Lingli

    2015-01-01

    Objective Elevated serum IgG4 levels are an important hallmark for diagnosing IgG4-related disease (IgG4-RD), but can also be observed in other diseases. This study aimed to compare two different testing methods for IgG4: ELISA and nephelometric assay. Both assays were used to measure serum IgG4 concentrations, and to assess the prevalence of high serum IgG4 levels in both IgG4-RD and non-IgG4-RD diseases. Methods A total of 80 serum samples were tested using the nephelometric assay and ELISA method that we established. Serum IgG4 concentrations were determined by ELISA for 957 patients with distinct diseases, including 12 cases of IgG4-RD and 945 cases of non-IgG4-RD. Results IgG4 levels from 80 selected serum samples examined by ELISA were in agreement with those detected using the nephelometry assay. Meanwhile, the serum IgG4 concentrations measured by ELISA were also consistent with the clinical diagnoses of patients with IgG4-RD during the course of disease. The Elevated levels of serum IgG4 (>1.35 g/L) were detected in all IgG4-RD (12/12) patients, and the prevalence of high IgG4 serum levels was 3.39% in non-IgG4-RD cases. Among them, the positive rates of serum IgG4 were 2.06% in patients with carcinoma and 6.3% in patients with other non-IgG4 autoimmune diseases. Conclusion Our established ELISA method is a reliable and convenient technique, which could be extensively used in the clinic to measure serum IgG4 levels. High levels of IgG4 were observed in IgG4-RD. However, this phenomenon could also be observed in other diseases, such as carcinomas and other autoimmune diseases. Thus, a diagnosis of IgG4 disease cannot only be dependent on the detection of elevated serum IgG4 levels. PMID:25885536

  10. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma

    DEFF Research Database (Denmark)

    Overdijk, M. B.; Verploegen, S.; Bogels, M.

    2015-01-01

    Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment...... in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly...... and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous...

  11. High prevalence of Toxoplasma gondii antibodies in dogs in Veracruz, Mexico

    Science.gov (United States)

    Little is known concerning the prevalence of Toxoplasma gondii infection in dogs in Mexico. Here, we investigated antibodies to T. gondii and associated risk factors in 101 dogs from an animal shelter in Veracruz State, Mexico. Canine sera were assayed for T. gondii IgG antibodies by using the modif...

  12. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  13. Influenza virus neutralizing antibodies and IgG isotype profiles after immunization of mice with influenza A subunit vaccine using various adjuvants

    NARCIS (Netherlands)

    Benne, CA; Harmsen, M; vanderGraaff, W; Verheul, AFM; Snippe, H; Kraaijeveld, CA

    The influence of various adjuvants on the development of influenza virus neutralizing antibodies and distribution of anti-influenza virus IgG isotypes after immunization of mice with influenza A (H3N2) subunit vaccine was investigated. Serum titres of influenza virus neutralizing antibodies and

  14. Anti-phosphatidylserine/prothrombin antibodies: an additional diagnostic marker for APS?

    Science.gov (United States)

    Pregnolato, Francesca; Chighizola, Cecilia B; Encabo, Susan; Shums, Zakera; Norman, Gary L; Tripodi, Armando; Chantarangkul, Veena; Bertero, Tiziana; De Micheli, Valeria; Borghi, Maria Orietta; Meroni, Pier Luigi

    2013-07-01

    Among the diagnostic assays for anti-phospholipid syndrome (APS), lupus anticoagulant (LA) is the strongest predictor of thrombosis; however, it presents several limitations as interference with anticoagulant therapy and poor inter-laboratory agreement. Two-thirds of LA activity is apparently due to antibodies against prothrombin (PT), usually detectable by ELISA. Binding of PT to phosphatidylserine (PS) has been shown to enhance solid-phase anti-PT assay sensitivity. To determine the prevalence of antibodies against PS/PT (aPS/PT) in APS, we tested the semiquantitative QUANTA Lite(®) aPS/PT ELISA in a cohort of 80 APS patients. The prevalence of aPS/PT was 81.3%, rising to 87.6% when considering LA-positive subjects only. We observed a strong correlation between aPS/PT and LA (p = 0.006). To note, APS patients with thrombotic manifestations displayed significantly higher IgG aPS/PT titers compared to 20 aPL asymptomatic carriers (p = 0.012). To rule out a possible cross-reactivity of anti-β2 glycoprotein I antibodies (aβ2GPI) with PS/PT complex, we tested two monoclonal aβ2GPI antibodies and an affinity-purified (AP) polyclonal aβ2GPI IgG obtained from the serum of a patient reacting against both β2GPI and PS/PT. The two monoclonal antibodies did not show any reactivity against PS/PT complex, similarly the AP IgGs did not react toward PS/PT antigen while preserved their aβ2GPI activity. Our findings suggest that aPS/PT are a definite antibody population in APS. Moreover, the good correlation between aPS/PT ELISA and LA may support its use as a surrogate test for LA, particularly useful to overcome the technical limitations of the functional assay.

  15. What is IgG4? A review of the biology of a unique immunoglobulin subtype.

    Science.gov (United States)

    Nirula, Ajay; Glaser, Scott M; Kalled, Susan L; Taylor, Frederick R; Taylora, Frederick R

    2011-01-01

    Recent descriptions of the group of clinical disorders collectively defined as IgG4-related systemic disease (IgG4-RSD) have prompted this review of the unique biology of the IgG4 antibody. This article will discuss IgG4 structure and function, the unique phenomenon of half-antibody exchange, and the implications of IgG4 biology for its proposed role in immunologic diseases. IgG4 antibodies have unique structural and functional properties and undergo 'half-antibody exchange' in vivo, resulting in recombined antibodies composed of two different binding specificities. The production of IgG4 antibodies appears to be driven in part by T helper 2 (Th2) cytokines that mediate allergic responses and IgE production. Although serum IgG4 levels in healthy individuals vary significantly, data from multiple sclerosis (MS) patients suggest tight regulation of individual IgG4 levels over time. IgG4-RSD represents a diverse group of clinical disorders unified by elevated IgG4 levels and specific histopathologic findings. A key unanswered question is whether IgG4, a relatively weak activator of effector cells, is pathogenic in these disorders. IgG4 is a unique antibody biologically and structurally. Increased understanding of its precise role in the clinical syndromes that comprise IgG4-RSD may ultimately elucidate the underlying pathogenesis.

  16. Effects of gamma rays on the immunogenicity (IgG types) of ovalbumin

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, J.A. E-mail: jbalves@ipen.br; Spencer, P.J.; Aroeira, L.G.S.; Casare, M.S.; Nascimento, N

    2004-10-01

    Ionizing radiation has been successfully employed to modify the immunological properties of biomolecules. Very promising results were obtained when crude animal venoms, as well as isolated toxins, were treated with gamma rays, yielding toxoids with good immunogenicity. However, little is known about the modifications that irradiated molecules undergo and even less about the immunological response that such antigens elicit. In the present work, we used ovalbumin as a model to investigate possible immunogenic differences between native and irradiated proteins. Native ovalbumin (2 mg/ml in 150 mM NaCl) was irradiated with 2 kGy of {sup 60}Co gamma rays with a 570 Gy/h dose rate. B10.PL mice (n=5) were then immunized with either the native or the irradiated protein. After three immunizations, serum samples were collected and the antibody titers and isotypes were determined by enzyme-linked immunoadsorbant assay. Our data indicate that no difference could be noticed when the antibody titers of the two groups were compared. However, the isotyping assays indicates that the native protein induced high levels of IgG1, while its irradiated counterpart displayed mostly IgG2b antibodies. These data suggest that after irradiation, an antigen known to induce a Th2 response, is able to switch the immune system towards a Th1 pattern.

  17. Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes.

    Science.gov (United States)

    Prodjinotho, Ulrich F; von Horn, Charlotte; Debrah, Alex Y; Batsa Debrah, Linda; Albers, Anna; Layland, Laura E; Hoerauf, Achim; Adjobimey, Tomabu

    2017-07-01

    Helminth parasites are known to be efficient modulators of their host's immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on

  18. Correlation between the Amount of Anti-D Antibodies and IgG Subclasses with Severity of Haemolytic Disease of Foetus and Newborn.

    Science.gov (United States)

    Velkova, Emilija

    2015-06-15

    The aim of this study was to investigate the influence of subclasses to IgG anti-D on the intensity of hemolytic disease of fetus and newborn (HDFN) at 45 fetuses/newborns with symptoms of mild and severe HDFN in Republic of Macedonia. In retrospective and prospective studies, in a period of 10 years, from 2004 to 2014, there have been immunohemathology tests performed on 22 009 samples on serums of pregnant women. At 37.78% of the total number of tested patients, IgG1 and IgG3 was the reason for severe HDFN. At 17.77% of the total number of tested patients, which had only IgG1detected, was the reason for serious intensity of HDFN. The correlation of the titer to anti-D antibodies in the mother's serum and the intensity of HDFN were researched in 48 newborns. The titers between 1:8 and 1:32 resulted in 3 cases of HDFN with symptoms of severe disease and in 4 cases there were no signs of HDFN. At 12 women that had a titre between 1:32 and 1:512, five of the newborns developed severe HDFN, and seven had symptoms of mild and weak intensity form. In 3 cases the titer was higher than 512, and out of them one newborn had weak symptoms of HDFN, one developed severe HDFN and one ended with foetal death. Only in one case the titer reached a value higher than 1000, and it ended with a fetal death. The titers of the pregnant women serum those are lower than 32 and those higher than 1000 can well predict HDFN. The titers of anti-D antibodies between 64 and 512 have no exact predictive value. IgG1 and IgG3 subclasses of anti-D have no predictive value by themselves, and cannot foresee the outcome of HDFN. The research study results suggest that IgG1 and IgG3 should be included in a multi - parameter protocol for evaluation of the HDFN intensity. They can give a real assessment of the expected HDFN intensity in combination with the titer hight and the significance of the antibodies.

  19. Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens

    DEFF Research Database (Denmark)

    Sparholt, S H; Barington, T; Schou, C

    1991-01-01

    Alutard, showed that 1 week after injection, circulating allergen-specific antibody-secreting cells (AbSC) were detected with this assay. The ranges were between 1 and 13 IgM-, 2 and 20 IgG- and 20 and 55 IgA allergen-specific AbSC per 10(6) MNC. It is expected that this assay will help to understand more...

  20. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    S Kaur

    2012-01-01

    Full Text Available Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P0.05 in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

  1. Association of beta2-glycoprotein I IgG and IgM antibodies with thrombosis and thrombocytopenia

    DEFF Research Database (Denmark)

    Voss, Anne-Sofie Boertmann; Jacobsen, Søren; Heegaard, Niels Henrik Helweg

    2001-01-01

    Antiphospholipid antibodies (APA) have been known for decades. Their relation to clinical manifestations, primarily thromboses and thrombocytopenia, was recognised in the 1980s. In this clinical study two cohorts of patients, a population-based (84 patients with systemic lupus erythematosus (SLE......)) and a hospital-based (87 patients with SLE and 53 with other connective tissue diseases) were investigated for APA and associated clinical manifestations. Anticardiolipin antibodies (ACA) of IgG and IgM classes were found in 13 and 38% of the population-based patients and in 29 and 58% of the hospital...

  2. Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2

    NARCIS (Netherlands)

    Yazdanbakhsh, M.; Paxton, W. A.; Brandenburg, A.; van Ree, R.; Lens, M.; Partono, F.; Maizels, R. M.; Selkirk, M. E.

    1995-01-01

    Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2

  3. A novel reporter system for neutralizing and enhancing antibody assay against dengue virus.

    Science.gov (United States)

    Song, Ke-Yu; Zhao, Hui; Jiang, Zhen-You; Li, Xiao-Feng; Deng, Yong-Qiang; Jiang, Tao; Zhu, Shun-Ya; Shi, Pei-Yong; Zhang, Bo; Zhang, Fu-Chun; Qin, E-De; Qin, Cheng-Feng

    2014-02-18

    Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.

  4. Parvovirus B19 antibodies and correlates of infection in pregnant women attending an antenatal clinic in central Nigeria

    Directory of Open Access Journals (Sweden)

    Samuel E Emiasegen

    2011-03-01

    Full Text Available Human parvovirus B19 infection is associated with spontaneous abortion, hydrops foetalis, intrauterine foetal death, erythema infectiosum (5th disease, aplastic crisis and acute symmetric polyarthropathy. However, data concerning Nigerian patients with B19 infection have not been published yet. The purpose of this study was to establish the prevalence of B19 IgG and IgM antibodies, including correlates of infection, among pregnant women attending an antenatal clinic in Nigeria. Subsequent to clearance from an ethical committee, blood samples were collected between August-November 2008 from 273 pregnant women between the ages of 15-40 years who have given their informed consent and completed self-administered questionnaires. Recombinant IgG and IgM enzyme linked immunosorbent assay kits (Demeditec Diagnostics, Germany were used for the assays. Out of the 273 participants, 111 (40.7% had either IgG or IgM antibodies. Out of these, 75 (27.5% had IgG antibodies whereas 36 (13.2% had IgM antibodies, and those aged 36-40 years had the highest prevalence of IgG antibodies. Significant determinants of infection (p < 0.05 included the receipt of a blood transfusion, occupation and the presence of a large number of children in the household. Our findings have important implications for transfusion and foeto-maternal health policy in Nigeria. Routine screening for B19 IgM antibodies and accompanying clinical management of positive cases should be made mandatory for all Nigerian blood donors and women of childbearing age.

  5. Sensitive radioimmunoassay for the determination of antibodies to mouse hepatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Leibowitz, J L [California Univ., San Diego, La Jolla (USA); Fung, L S; Levy, G A [Toronto Univ., Ontario (Canada)

    1983-05-01

    A solid-phase radioimmunoassay is described for the detection of antibodies to mouse hepatitis virus. Viruses were purified by velocity and isopycnic gradient centrifugation and 96-well plastic plates were coated with viral antigens. To allow the detection of most serotypes of low titered antisera, a pool of antigens from several viral serotypes were employed. The second antibody, an affinity-purified goat antimouse immunoglobulin, detects IgG, IgM and IgA antibodies. This assay is more sensitive than either the plaque reduction assay or the commercially available enzyme-linked immunosorbant assay and proved to be useful for screening mouse colonies for the presence of mouse hepatitis virus, following seroconversion in experimental animals and in the production of monoclonal antibodies to both structural and nonstructural proteins.

  6. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  7. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Directory of Open Access Journals (Sweden)

    Michael A. Partridge

    2016-01-01

    Full Text Available Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.

  8. Role of anti-domain 1-β2 glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome.

    Science.gov (United States)

    De Craemer, A-S; Musial, J; Devreese, K M J

    2016-09-01

    Essentials Antibodies to domain 1 of β2 glycoprotein I (aD1) are a subset of antiphospholipid antibodies. We evaluated the added diagnostic value of an automated aD1 assay in antiphospholipid syndrome. AD1 IgG correctly classifies patients at risk for thrombosis. Agreement between aD1 and aβ2GPI IgG is high, limiting the added value of aD1 in our setting. Click to hear Professor de Groot's perspective on new mechanistic understanding in antiphospholipid syndrome Background Laboratory diagnosis of antiphospholipid syndrome (APS) includes lupus anticoagulant (LAC), anticardiolipin (aCL) or anti-β2 glycoprotein I (aβ2 GPI) antibodies. Antibodies targeting domain 1 of β2 GPI (aD1) constitute a pathogenic subset of autoantibodies. Objectives In this cohort study, we determined the clinical performance characteristics, additional diagnostic value and the contribution to APS risk stratification of an automated aD1 assay. Patients/Methods LAC, aCL, aβ2 GPI and aD1 IgG were measured in 101 APS patients, 123 patients with autoimmune disorders, 82 diseased controls and 120 healthy controls. aD1 antibodies were detected by QUANTA Flash(®) Beta2GPI-Domain 1 chemiluminescence immunoassay. Results With a cut-off value of 20.0 CU, the aD1 IgG assay identifies APS patients in a clinically affected patient cohort with a sensitivity of 53.5% and specificity of 98.8%. It implied a high odds ratio (OR) for clinical events (OR, 17.0; 95% confidence interval [CI], 7.1-40.5). aD1 IgG did not add diagnostic value to the formal aPL panel because aβ2 GPI IgG was nearly as specific but more sensitive for APS (sensitivity 56.4%) with a higher OR for clinical events (36.2; 95% CI, 11.1-117.9). High aD1 titers identify triple-positive patients and patients with thrombosis in a β2 GPI-dependent LAC-positive population. Agreement between aD1 IgG and aβ2 GPI IgG was high (positive and negative agreement 91.7% and 98.4%, respectively). Conclusion Detection of aD1 IgG correctly classifies

  9. Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

    Directory of Open Access Journals (Sweden)

    AM Marassá

    2009-01-01

    Full Text Available With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA. A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027 in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

  10. Seropositivity and determinants of immunoglobulin-G (IgG ...

    African Journals Online (AJOL)

    Background: This study is the first documented prevalence of IgG antibody against HSV-1&-2 in Port Harcourt, Nigeria and thus provides baseline data for future in-depth studies on HSV infection in South-South, Nigeria. Objective: This study determined the seropositivity and determinants of serum IgG antibody against ...

  11. A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

    International Nuclear Information System (INIS)

    Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

    1982-01-01

    A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

  12. A solid-phase radioimmunoassay for IgG gliadin antibodies using 125I-labelled staphylococcal protein A

    International Nuclear Information System (INIS)

    Troncone, R.; Pignata, C.; Farris, E.; Ciccimarra, F.

    1983-01-01

    A sensitive radioimmunoassay for IgG gliadin antibodies is described. Serum specimens were added to wells of plastic microtitre plates coated with gliadin. After removal of the unbound material, gliadin antibodies were detected by adding 125 I-labelled staphylococcal protein A ( 125 I-SpA). Serum specimens from coeliac patients on a normal diet or on a gluten-free diet were tested, as well as sera from an age-matched control group. Measurements to obtain precise quantitative values were made with gliadin antibody-rich serum as reference standard. High titres of gliadin antibodies were found in 18 out of 19 coeliac patients on a normal diet (95%); in patients on a strict gluten-free diet serum values did not exceed 2 S.D. of the control mean. Due to the high sensitivity of the method a low but detectable amount of gliadin antibody was present in the sera of all controls. (Auth.)

  13. Solid-phase radioimmunoassay for IgG gliadin antibodies using /sup 125/I-labelled staphylococcal protein A

    Energy Technology Data Exchange (ETDEWEB)

    Troncone, R.; Pignata, C.; Farris, E.; Ciccimarra, F. (Naples Univ. (Italy). II Facolta di Medicina)

    1983-10-14

    A sensitive radioimmunoassay for IgG gliadin antibodies is described. Serum specimens were added to wells of plastic microtitre plates coated with gliadin. After removal of the unbound material, gliadin antibodies were detected by adding /sup 125/I-labelled staphylococcal protein A (/sup 125/I-SpA). Serum specimens from coeliac patients on a normal diet or on a gluten-free diet were tested, as well as sera from an age-matched control group. Measurements to obtain precise quantitative values were made with gliadin antibody-rich serum as reference standard. High titres of gliadin antibodies were found in 18 out of 19 coeliac patients on a normal diet (95%); in patients on a strict gluten-free diet serum values did not exceed 2 S.D. of the control mean. Due to the high sensitivity of the method a low but detectable amount of gliadin antibody was present in the sera of all controls.

  14. Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro*

    Science.gov (United States)

    Liu, Ling; Jacobsen, Frederick W.; Everds, Nancy; Zhuang, Yao; Yu, Yan Bin; Li, Nianyu; Clark, Darcey; Nguyen, Mai Phuong; Fort, Madeline; Narayanan, Padma; Kim, Kei; Stevenson, Riki; Narhi, Linda; Gunasekaran, Kannan; Bussiere, Jeanine L.

    2017-01-01

    The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem. 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro. These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions. PMID:27994063

  15. A sensitive radioimmunoassay for the determination of antibodies to mouse hepatitis virus

    International Nuclear Information System (INIS)

    Leibowitz, J.L.; Fung, L.S.; Levy, G.A.

    1983-01-01

    A solid-phase radioimmunoassay is described for the detection of antibodies to mouse hepatitis virus. Viruses were purified by velocity and isopycnic gradient centrifugation and 96-well plastic plates were coated with viral antigens. To allow the detection of most serotypes of low titered antisera, a pool of antigens from several viral serotypes were employed. The second antibody, an affinity-purified goat antimouse immunoglobulin, detects IgG, IgM and IgA antibodies. This assay is more sensitive than either the plaque reduction assay or the commercially available enzyme-linked immunosorbant assay and proved to be useful for screening mouse colonies for the presence of mouse hepatitis virus, following seroconversion in experimental animals and in the production of monoclonal antibodies to both structural and nonstructural proteins. (Auth.)

  16. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    Science.gov (United States)

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

    2013-01-01

    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  17. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    Science.gov (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  18. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao

    2015-01-01

    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  19. Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Yannic C. Bartsch

    2018-06-01

    Full Text Available Pro- and anti-inflammatory effector functions of IgG antibodies (Abs depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0 IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors, administered in high doses (2 g/kg to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.

  20. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy.

    Science.gov (United States)

    Hébert, J; Bernier, D; Mourad, W

    1990-06-01

    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  1. Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

    Science.gov (United States)

    Sun, Xiao-Jing; Chen, Min; Zhao, Ming-Hui

    2018-03-01

    Cumulating evidences suggested an important role of sphingosine-1-phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)-ANCA-positive IgG-mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO-ANCA-positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane-bound and soluble ICAM-1 and VCAM-1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO-ANCA-positive IgG-induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO-ANCA-positive IgG. S1P enhanced MPO-ANCA-positive IgG-induced membrane-bound and soluble ICAM-1/VCAM-1 up-regulation of GEnCs. Soluble ICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG increased upon pre-incubation of S1PR1 antagonist, while pre-incubation of GEnCs with the S1PR1 agonist down-regulated sICAM-1 level. Blocking S1PR2-4 reduced sICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG. Pre-incubation with S1PR5 agonist could increase sICAM-1 level in the supernatants of GEnC stimulated by S1P and MPO-ANCA-positive IgG. S1P can enhance MPO-ANCA-positive IgG-mediated GEnC activation through S1PR2-5. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Antibodies Against Hypocretin Receptor 2 Are Rare in Narcolepsy.

    Science.gov (United States)

    Giannoccaro, Maria Pia; Waters, Patrick; Pizza, Fabio; Liguori, Rocco; Plazzi, Giuseppe; Vincent, Angela

    2017-02-01

    Recently, antibodies to the hypocretin receptor 2 (HCRTR2-Abs) were reported in a high proportion of narcolepsy patients who developed the disease following Pandemrix® vaccination. We tested a group of narcolepsy patients for the HCRTR2-Abs using a newly established cell-based assay. Sera from 50 narcolepsy type 1 (NT1) and 11 narcolepsy type 2 (NT2) patients, 22 patients with other sleep disorders, 15 healthy controls, and 93 disease controls were studied. Cerebrospinal fluid (CSFs) from three narcoleptic patients were subsequently included. Human embryonic kidney cells were transiently transfected with human HCRTR2, incubated with patients' sera for 1 hr at 1:20 dilution and then fixed. Binding of antibodies was detected by fluorescently labeled secondary antibodies to human immunoglobulin G (IgG) and the different IgG subclasses. A nonlinear visual scoring system was used from 0 to 4; samples scoring ≥1 were considered positive. Only 3 (5%) of 61 patients showed a score ≥1, one with IgG1- and two with IgG3-antibodies, but titers were low (1:40-1:100). CSFs from these patients were negative. The three positive patients included one NT1 case with associated psychotic features, one NT2 patient, and an NT1 patient with normal hypocretin CSF levels. Low levels of IgG1 or IgG3 antibodies against HCRTR2 were found in 3 of 61 patients with narcolepsy, although only 1 presented with full-blown NT1. HCRTR2-Abs are not common in narcolepsy unrelated to vaccination. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  3. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    Science.gov (United States)

    Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

  4. Bronchial asthma is not associated with auto-antibodies to lipocortin-1.

    Science.gov (United States)

    Wilkinson, J R; Podgorski, M R; Godolphin, J L; Goulding, N J; Lee, T H

    1990-03-01

    Corticosteroids may mediate some of their anti-inflammatory action by the induction of lipocortin-1, which inhibits phospholipase A2 activity. Raised levels of antibodies to lipocortin have been found in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and it has been postulated that these may contribute to steroid resistance. A proportion of asthmatic patients fail to respond to treatment with corticosteroids and one possible mechanism is that these patients have raised levels of anti-lipocortin antibodies. We have therefore measured IgG and IgM antibodies to lipocortin by enzyme linked immunosorbent assay (ELISA) in eight corticosteroid-sensitive (CS) and 7 corticosteroid-resistant (CR) asthmatic subjects, and in eight normal controls. Comparison of asthmatic subjects with normal controls revealed no significant differences in either IgG or IgM antibodies to lipocortin. Comparison of CS asthmatic subjects with CR asthmatic subjects similarly revealed no significant differences in the concentration of either IgG or IgM antibodies to lipocortin. Levels of anti-lipocortin antibodies did not correlate with clinical response to treatment with 40 mg/day of prednisolone. Anti-lipocortin antibodies are unlikely to be involved in the inflammation seen in asthma, or in the relative insensitivity to corticosteroids seen in CR asthmatic subjects.

  5. Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

    Science.gov (United States)

    Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

    2017-09-05

    An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Seroprevalence of Toxoplasma gondii IgG antibody in HIV/AIDS-infected individuals in Maputo, Mozambique

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    Abílio Domingos

    2013-10-01

    Full Text Available OBJECTIVE To analyze the prevalence of IgG antibodies to Toxoplasma gondii in patients infected with HIV/AIDS and the association of demographic and social variables. METHODS Descriptive cross-sectional study that included the analysis of sociodemographic data and laboratory findings of 200 patients infected with HIV/AIDS treated in a laboratory unit in Maputo, Mozambique, in 2010. Individual data for all participants were collected with a self-administered questionnaire. Plasma samples were tested for IgG testing of anti- T. gondii using hemagglutination for the analysis of antibodies. RESULTS The seroprevalence of IgG anti- T. gondii was 46.0% (95%CI 39.2;52.9, 39.3% (95%CI 29.5;50.0 in men and 50.9% (95%CI 41.9;59.8 in women, with no difference between sex (OR 1.30; 95%CI 0.95;1.77; p = 0.12. Ages ranged from 10 to 60 years, with a higher prevalence of infection in older age groups, but with no significant difference between them. Regularly consuming cattle meat (OR 1.74; 95%CI 1.04;2.89, p = 0.05, breeding cats/dogs (OR 6.18; 95%CI 3.60;10.62, p < 0.000 and having regular contact with soil (OR 3.38; 95%CI 2.19;5.21; p < 0.000 were significantly associated with risk of latent infection. CONCLUSIONS Toxoplasmosis is an infection with high prevalence in Mozambique. Cultural and behavioral aspects increase the risk. Toxoplasmosis can be responsible in our environment by the great burden of morbidity and mortality associated with meningoencephalic injuries in patients with HIV/AIDS.

  7. Perfil de isotipos de imunoglobulinas e subclasses de IgG na leishmaniose tegumentar americana Immunoglobulin isotype and IgG subclass profiles in american tegumentary leishmaniasis

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    Maria Aparecida de Souza

    2005-04-01

    Full Text Available O presente trabalho avaliou o perfil de anticorpos em amostras de soro de 37 pacientes com diagnóstico clínico confirmado ou compatível com leishmaniose tegumentar americana atendidos no Hospital de Clínicas da Universidade Federal de Uberlândia, MG. Os perfis das classes de imunoglobulinas e subclasses de IgG foram analisados pelo teste ELISA indireto, utilizando-se antígeno solúvel de Leishmania (Leishmania amazonensis. A avidez dos anticorpos foi determinada pelo tratamento com uréia a 6 M, após incubação dos soros com o antígeno. Observou-se que 97%, 94,6%, 57,5 e 21,5% das amostras testadas apresentaram anticorpos anti-Leishmania das classes IgE, IgG, IgA e IgM, respectivamente e, os perfis das subclasses de IgG demonstraram, IgG1>IgG3>IgG2>IgG4. Os anticorpos IgE anti-Leishmania de alta avidez corresponderam a 44,4%. Por outro lado, IgG e IgA anti-Leishmania foram em sua maioria (62,8 e 47,8%, respectivamente, de média avidez. A variação do perfil de isotipos, bem como a avidez das imunoglobulinas refletiu a complexidade da resposta imune humoral contra a leishmaniose tegumentar americana.The present work investigated the serum antibody profiles in 37 patients with American tegumentary leishmaniasis, who were attended at Hospital de Clinicas - Universidade Federal de Uberlandia, MG, Brazil. The immunoglobulin class and IgG subclass profiles were analyzed by indirect ELISA using Leishmania (Leishmania amazonensis soluble antigen. The antibody avidity was determined by 6 M urea treatment after incubation with immunoenzymatic conjugate. It was observed that 97% of the serum samples presented anti-Leishmania antibodies for IgE class, 94.6% IgG, 57.5% IgA and 21.5% IgM class. For IgG subclasses the profiles were in the following order of frequency: IgG1>IgG3>IgG2>IgG4. High avidity of anti-Leishmania IgE antibodies was found in 44.4% of the samples. On the other hand, moderate avidity of specific IgG and IgA was observed in 62

  8. Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

    Science.gov (United States)

    Chen, Chang-Hsin; Abi-Ghanem, Daad; Waghela, Suryakant D; Chou, Wen-Ko; Farnell, Morgan B; Mwangi, Waithaka; Berghman, Luc R

    2012-04-30

    Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Thrombosis and antiphospholipid antibody syndrome during acute Q fever

    Science.gov (United States)

    Million, Matthieu; Bardin, Nathalie; Bessis, Simon; Nouiakh, Nadia; Douliery, Charlaine; Edouard, Sophie; Angelakis, Emmanouil; Bosseray, Annick; Epaulard, Olivier; Branger, Stéphanie; Chaudier, Bernard; Blanc-Laserre, Karine; Ferreira-Maldent, Nicole; Demonchy, Elisa; Roblot, France; Reynes, Jacques; Djossou, Felix; Protopopescu, Camelia; Carrieri, Patrizia; Camoin-Jau, Laurence; Mege, Jean-Louis; Raoult, Didier

    2017-01-01

    Abstract Q fever is a neglected and potentially fatal disease. During acute Q fever, antiphospholipid antibodies are very prevalent and have been associated with fever, thrombocytopenia, acquired heart valve disease, and progression to chronic endocarditis. However, thrombosis, the main clinical criterion of the 2006 updated classification of the antiphospholipid syndrome, has not been assessed in this context. To test whether thrombosis is associated with antiphospholipid antibodies and whether the criteria for antiphospholipid syndrome can be met in patients with acute Q fever, we conducted a cross-sectional study at the French National Referral Center for Q fever. Patients included were diagnosed with acute Q fever in our Center between January 2007 and December 2015. Each patient's history and clinical characteristics were recorded with a standardized questionnaire. Predictive factors associated with thrombosis were assessed using a rare events logistic regression model. IgG anticardiolipin antibodies (IgG aCL) assessed by an enzyme-linked immunosorbent assay were tested on the Q fever diagnostic serum. A dose-dependent relationship between IgG aCL levels and thrombosis was tested using a receiver operating characteristic (ROC) analysis. Of the 664 patients identified for inclusion in the study, 313 (47.1%) had positive IgG aCL and 13 (1.9%) were diagnosed with thrombosis. Three patients fulfilled the antiphospholipid syndrome criteria. After multiple adjustments, only positive IgG aCL (relative risk, 14.46 [1.85–113.14], P = .011) were independently associated with thrombosis. ROC analysis identified a dose-dependent relationship between IgG aCL levels and occurrence of thrombosis (area under curve, 0.83, 95%CI [0.73–0.93], P antiphospholipid antibodies are associated with thrombosis, thrombocytopenia, and acquired valvular heart disease. Antiphospholipid antibodies should be systematically assessed in acute Q fever patients. Hydroxychloroquine

  10. Aggregation and pH-temperature phase behavior for aggregates of an IgG2 antibody.

    Science.gov (United States)

    Sahin, Erinc; Weiss, William F; Kroetsch, Andrew M; King, Kevin R; Kessler, R Kendall; Das, Tapan K; Roberts, Christopher J

    2012-05-01

    Monomer unfolding and thermally accelerated aggregation kinetics to produce soluble oligomers or insoluble macroscopic aggregates were characterized as a function of pH for an IgG2 antibody using differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC). Aggregate size was quantified via laser light scattering, and aggregate solubility via turbidity and visual inspection. Interestingly, nonnative oligomers were soluble at pH 5.5 above approximately 15°C, but converted reversibly to visible/insoluble particles at lower temperatures. Lower pH values yielded only soluble aggregates, whereas higher pH resulted in insoluble aggregates, regardless of the solution temperature. Unlike the growing body of literature that supports the three-endotherm model of IgG1 unfolding in DSC, the results here also illustrate limitations of that model for other monoclonal antibodies. Comparison of DSC with monomer loss (via SEC) from samples during thermal scanning indicates that the least conformationally stable domain is not the most aggregation prone, and that a number of the domains remain intact within the constituent monomers of the resulting aggregates. This highlights continued challenges with predicting a priori which domain(s) or thermal transition(s) is(are) most relevant for product stability with respect to aggregation. Copyright © 2012 Wiley Periodicals, Inc.

  11. Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy.

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    Kathrin Doppler

    Full Text Available Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients' sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy.

  12. Effect of IgG subclasses on in vivo bioavailability and metabolic fate of immune-complexed insulin in Lewis rats

    International Nuclear Information System (INIS)

    Arquilla, E.R.; Stenger, D.; McDougall, B.; Ulich, T.R.

    1987-01-01

    The bioavailability, distribution, and metabolic fate of 125 I-labeled insulin complexed to antibodies in guinea pig antiserum, purified guinea pig IgG1, IgG2, a mixture of IgG1 and IgG2, and homologous Lou/m rat antiserum were studied in inbred Lewis rats. 125 I-insulin complexed to purified guinea pig IgG2 antibodies was rapidly cleared from the blood and sequestered in increasing amounts with time in the liver. Large amounts of the 125 I-insulin complexed to guinea pig IgG1 antibodies remained in the blood for at least 30 min. The bioavailability of 125 I-insulin bound to IgG1 and IgG2 antibodies was inhibited for at least 30 min because significantly less was available for rapid binding to insulin receptors on hepatocytes and renal tubular cells and its subsequent rapid degradation. The bioavailability of 125 I-insulin was further decreased when bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies compared with the 125 I-insulin complexed to either purified IgG1 or IgG2 antibodies alone. The 125 I-insulin bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies was distributed in vivo in a manner reflecting the relative concentrations of the IgG1 and IgG2 antibodies present. The bioavailability, distribution, and metabolic fate of 125 I-insulin in immune complexes prepared with homologous Lou/m rat insulin antiserum was qualitatively similar to that observed with immune complexes prepared with guinea pig insulin antiserum. It appears that the Lewis rat can be used as an in vivo model to study the bioavailability,distribution,and metabolic fate of insulin bound to xenogenic or homologous insulin antibodies

  13. Solid-phase radioimmunoassay for the detection fo IgG antibodies to gramineae pollen (Dactylis glomerata) and to acarida (dermatophagoides Pteronyssinus)

    Energy Technology Data Exchange (ETDEWEB)

    Guilloux, L.; Ville, G.

    1985-01-01

    A solid-phase radioimmunoassay using SVI-protein A onto polystyrene microplates, was applied successfully for the detection of IgG antibodies to acarida and to grass pollen in allergic patients and in patients undergoing desensitization therapy.

  14. Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2012-01-01

    Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

  15. Trends in the patterns of IgM and IgG antibodies in febrile persons with suspected dengue in Barbados, an English-speaking Caribbean country, 2006–2013

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    Alok Kumar

    2015-11-01

    Full Text Available Summary: Long-term seroprevalence studies of dengue have provided a measure of the degree of endemicity and future trends in disease prevalence and severity. In this study, we describe the seroprevalence of dengue antibodies in febrile persons with suspected acute dengue in Barbados. It is a retrospective population-based study of all febrile persons with suspected dengue from 2006 to 2013. All of the cases had IgM and IgG antibodies in the blood sample drawn between days 3 and 5 of their illness. Among the 8296 cases that were tested for IgM antibodies, 3037 (36.6% had recent dengue infection. In the age groups 20 years, 23.3%, 39.6% and 35.5% had acute infection, respectively. Of the 7227 cases with documented IgG results, 5473 (75.7% were positive and had a past infection. In the age groups 20 years, 31.2%, 65.2% and 86.6%, respectively, had a past infection (IgG positive. During the first 5 years of life, 10–20% of febrile persons investigated for dengue had a positive IgM and a negative IgG titer, between 5 and 10% had a positive IgM and IgG titer, 5% had a positive IgG and a negative IgM titer, and between 45% and 65% had a negative IgM and a negative IgG titer. Throughout the study period, between 12% and 20% of febrile persons failed to show any evidence of current or previous dengue. In the age groups 20 years, 45.0%, 18.8% and 7.2%, respectively, had no evidence of recent or past dengue (both IgM and IgG negative. Between 37% and 59% of the febrile persons had serological evidence of past dengue in the absence of any current dengue. In conclusion, the pattern of IgG antibodies in this study was comparable to those in countries known to be hyperendemic for dengue. The age of infection is likely to shift to younger adults and children who are more likely to have severe dengue in the future. Keywords: Dengue, Seroprevalence, Febrile, Caribbean

  16. Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

    Science.gov (United States)

    Chung, Amy; Makuba, Jenniffer M; Ndlovu, Bongiwe; Licht, Anna; Robinson, Hannah; Ramlakhan, Yathisha; Ghebremichael, Musie; Reddy, Tarylee; Goulder, Philip; Walker, Bruce; Ndung'u, Thumbi; Alter, Galit

    2018-04-03

    Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naïve participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4 counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

  17. Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis

    NARCIS (Netherlands)

    Kurniawan, A.; Yazdanbakhsh, M.; van Ree, R.; Aalberse, R.; Selkirk, M. E.; Partono, F.; Maizels, R. M.

    1993-01-01

    A population of 164 adult individuals resident in an area endemic for Brugia malayi lymphatic filariasis has been studied for humoral immune responses to filarial parasites. Antibody levels to Ag extracted from adult worms were determined for each of the IgG subclasses, for IgM and for IgE. The

  18. Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability.

    Science.gov (United States)

    Peters, Shirley J; Smales, C Mark; Henry, Alistair J; Stephens, Paul E; West, Shauna; Humphreys, David P

    2012-07-13

    The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

  19. Solid-phase radioimmunoassay for the detection fo IgG antibodies to gramineae pollen (Dactylis glomerata) and to acarida (dermatophagoides Pteronyssinus)

    International Nuclear Information System (INIS)

    Guilloux, L.; Ville, G.

    1985-01-01

    A solid-phase radioimmunoassay using 125 I-protein A onto polystyrene microplates, was applied successfully for the detection of IgG antibodies to acarida and to grass pollen in allergic patients and in patients undergoing desensitization therapy [fr

  20. Preparación de un conjugado peroxidasa-anti IgG humana (cadena en conejo Preparation of a peroxidase-anti human IgG conjugate (chain g in rabbit

    Directory of Open Access Journals (Sweden)

    Julio C Merlín Linares

    2001-08-01

    Full Text Available Se preparó un conjugado con peroxidasa a partir de los anticuerpos específicos aislados de un suero de conejo anti cadenas g de la IgG humana. Los anticuerpos específicos se aislaron por cromatografía de afinidad, y el conjugado se preparó por el método de oxidación con peryodato. El conjugado obtenido presentó una relación molar IgG/peroxidasa de 1,07, un valor de RZ de 0,33 y resultó evaluado satisfactoriamente en cuanto a su especificidad y reactividad en los ensayos inmunoenzimáticos realizadosA peroxidase conjugate was prepared starting from the specific antibodies isolated from an anti-chain rabbit serum and from human IgG. The specific antibodies were isolated by affinity chromatography and the conjugate was prepared by the method of oxidation with periodate. The conjugate obtained presented a molar IgG/peroxidase relation of 1.07, a RZ value of 0.33 and it was satisfactorily evaluated as regards its specificity and reactivity in the immunoenzimatic assays carried out

  1. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    Science.gov (United States)

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  2. Measurement of the IgG2 response to Pneumococcal capsular polysaccharides may identify an antibody deficiency in individuals referred for immunological investigation.

    Science.gov (United States)

    Parker, Antony; Irure Ventura, Juan; Sims, Dawn; Echeverría de Carlos, Ainara; Gómez de la Torre, Ricardo; Tricas Aizpún, Lourdes; Ocejo-Vinyals, J Gonzalo; López-Hoyos, Marcos; Wallis, Gregg; Harding, Stephen

    2017-01-01

    IgG2 is the most efficient subclass for providing protection against pneumococcal pathogens. We hypothesised that some individuals may be unable to mount an effective pneumococcal capsular polysaccharide (PCP) IgG2 response despite having a normal PCP IgG concentration (PCP IgG2 deficient). The median pre-vaccination PCP IgG2 concentration was significantly lower in individuals referred for immunological investigation compared to healthy controls (2.8 mg/L range, 95% CI 1.1-88 vs. 29.5mg/L, 95% CI 13.5-90, p = 0.0002). PCP IgG:IgG2 ratios were significantly higher for the referral population than for healthy controls suggesting the increased production of PCP specific subclasses other than IgG2. The percentage of individuals with PCP IgG2 deficiency was significantly higher in referral groups compared to controls (31% vs. 5%; p = 0.0009) and in an individual with PCP IgG2 deficiency, the balance of PCP specific IgG subclass antibodies post vaccination changed from IgG2>IgG1>IgG3>IgG4 to IgG1>IgG3>IgG2>IgG4. The median PCP IgG2 concentration in those with PCP IgG2 deficiency was significantly lower in the referral groups compared to controls (7.8 mg/L, 95% CI 1.1-12 vs. 12.7 mg/L, 95% CI 11.8-13.1; p = 0.006). The data suggests a defect in the production PCP IgG2 may be present in individuals with normal PCP IgG referred for immunological investigation.

  3. Serum IgG antibody levels to periodontal microbiota are associated with incident Alzheimer disease.

    Directory of Open Access Journals (Sweden)

    James M Noble

    Full Text Available Periodontitis and Alzheimer disease (AD are associated with systemic inflammation. This research studied serum IgG to periodontal microbiota as possible predictors of incident AD.Using a case-cohort study design, 219 subjects (110 incident AD cases and 109 controls without incident cognitive impairment at last follow-up, matched on race-ethnicity, were drawn from the Washington Heights-Inwood Columbia Aging Project (WHICAP, a cohort of longitudinally followed northern Manhattan residents aged >65 years. Mean follow-up was five years (SD 2.6. In baseline sera, serum IgG levels were determined for bacteria known to be positively or negatively associated with periodontitis (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans Y4, Treponema denticola, Campylobacter rectus, Eubacterium nodatum, and Actinomyces naeslundii genospecies-2. In all analyses, we used antibody threshold levels shown to correlate with presence of moderate-severe periodontitis.Mean age was 72 years (SD 6.9 for controls, and 79 years (SD 4.6 for cases (p640 ng/ml, present in 10% of subjects was associated with increased risk of AD (HR = 2.0, 95%CI: 1.1-3.8. This association was stronger after adjusting for other significant titers (HR = 3.1, 95%CI: 1.5-6.4. In this model, high anti-E. nodatum IgG (>1755 ng/ml; 19% of subjects was associated with lower risk of AD (HR = 0.5, 95%CI: 0.2-0.9.Serum IgG levels to common periodontal microbiota are associated with risk for developing incident AD.

  4. Presence of anti-Toxoplasma antibodies in humans and their cats in the urban zone of Guadalajara

    Directory of Open Access Journals (Sweden)

    Galván Ramírez María de la Luz

    1999-01-01

    Full Text Available Cats are the definitive hosts of Toxoplasma gondii. Infected cats excrete oocysts in their feces, infecting humans and other animals. The objective of the present study was to determine the presence of anti-Toxoplasma antibodies in cat owners and their pets, and determine if there was a relationship between Toxoplasma infection and humans who live with infected cats. IgG anti-Toxoplasma antibodies in sera of 59 cat owners were determined by enzyme-linked immunosorbent assay (ELISA, in 24 sera from their cats, IgG, IgM, and IgA antibodies were found using Burney's ELISA. Thirty-eight (64% of 59 cat owners were positive to IgG anti-Toxoplasma. Seropositivity for cats was 70.8% IgG, 8.3% IgM, and 62.5% IgA. Cohabitation with cats infected by T. gondii, feeding with leftovers or raw viscera, and lack of control over how their feces were handled are risk factors conducive for humans to become infected by T. gondii.

  5. Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

    Science.gov (United States)

    Reyes, L; Hartikka, J; Bozoukova, V; Sukhu, L; Nishioka, W; Singh, G; Ferrari, M; Enas, J; Wheeler, C J; Manthorpe, M; Wloch, M K

    2001-06-14

    Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

  6. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  7. A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

    OpenAIRE

    Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

    2016-01-01

    Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

  8. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C

    2000-01-01

    of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  9. Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

    Science.gov (United States)

    Daley, L P; Gagliardo, L F; Duffy, M S; Smith, M C; Appleton, J A

    2005-03-01

    Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

  10. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...

  11. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

    NARCIS (Netherlands)

    Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

    2003-01-01

    To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

  12. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    Directory of Open Access Journals (Sweden)

    Yanil R Parma

    2012-06-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC, a subset of Shiga toxin producing E. coli (STEC is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic uremic syndrome (HUS. Regardless of serotype, Shiga toxins (Stx1 and/or Stx2 are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx was developed using anti-Stx2 B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933 and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 400 ng /ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for 2 strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

  13. Simultaneous use of serum IgG and IgM for risk scoring of suspected early Lyme borreliosis: graphical and bivariate analyses

    DEFF Research Database (Denmark)

    Dessau, Ram B; Ejlertsen, Tove; Hilden, Jørgen

    2010-01-01

    The laboratory diagnosis of early disseminated Lyme borreliosis (LB) rests on IgM and IgG antibodies in serum. The purpose of this study was to refine the statistical interpretation of IgM and IgG by combining the diagnostic evidence provided by the two immunoglobulins and exploiting the whole...... range of the quantitative variation in test values. ELISA assays based on purified flagella antigen were performed on sera from 815 healthy Danish blood donors as negative controls and 117 consecutive patients with confirmed neuroborreliosis (NB). A logistic regression model combining the standardized...... units of the IgM and IgG ELISA assays was constructed and the resulting disease risks graphically evaluated by receiver operating characteristic and 'predictiveness' curves. The combined model improves the discrimination between NB patients and blood donors. Hence, it is possible to report a predicted...

  14. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  15. PREVALENCE OF ANTIBODIES TO HUMAN PARVOVIRUS B19 IN SAUDI WOMEN OF CHILDBEARING AGE IN MAKKAH

    Science.gov (United States)

    Ghazi, Hani O.

    2007-01-01

    Objectives: To determine the seroprevalence rate of immunoglobulin G (IgG) and immunoglobulin M (IgM) to parvovirus B19 in pregnant Saudi women in Makkah. Subjects and Methods: Using enzyme-linked immunosorbent assay (ELISA), a total of 1200 serum samples were tested for antibodies to parvovirus B19 known to cause a variety of clinical syndromes in women and newborn infants. Results: Parvovirus B19 IgG antibodies detected in 46.6% and IgM antibodies were found in 2.25% of different age groups. Conclusion: The previous exposure to parvovirus B19 was determined, and 560 (46.6%) of 1200 pregnant Saudi women tested at their first antenatal visit were seropositive for specific IgG. The rate of maternal infection in susceptible pregnancies was 2.25%. These results were in accordance with previous studies performed in other countries. PMID:23012138

  16. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

    Science.gov (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

    2008-08-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  17. Detection of phase I IgG antibodies to Coxiella burnetii with EIA as a screening test for blood donations

    NARCIS (Netherlands)

    van der Hoek, W.; Wielders, C. C. H.; Schimmer, B.; Wegdam-Blans, M. C. A.; Meekelenkamp, J.; Zaaijer, H. L.; Schneeberger, P. M.

    2012-01-01

    The presence of a high phase I IgG antibody titre may indicate chronic infection and a risk for the transmission of Coxiella burnetii through blood transfusion. The outbreak of Q fever in the Netherlands allowed for the comparison of an enzyme immunoassay (EIA) with the reference immunofluorescence

  18. Limitations of a hemolytic plaque assay for IgG-anti-IgG rheumatoid factor-producing cells.

    Science.gov (United States)

    Venn, A J; Dresser, D W

    1987-09-24

    An attempt has been made to develop a hemolytic plaque assay capable of detecting homophile IgG rheumatoid factor (RF)-producing cells. Anti-immunoglobulin allotype-developing reagents were used to distinguish between target and effector IgG. The hemolytic assay has been used to demonstrate an apparently high level of homophile IgM and IgG RF-producing cells in the spleens and lymph nodes of mice stimulated by LPS. However, it appears that a large proportion of the plaques obtained in these assays are due to an artefact resulting from cross-linking of target and effector molecules by the developing reagents. In the case of IgM RF the artefact depends on the presence of a small contamination of the target IgG by IgM, allowing cross-linking of target and effector IgM by the anti-mu-specific developing reagent. With the IgG RF, cross-reactivity of the rabbit anti-Ighb allotype-developing serum for the 'wrong' (Igha) allotype, normally undetectable, becomes sufficient to be biologically relevant when the developing antibody is complexed by being bound to its target (Ighb) allotype. Nevertheless anti-allotype reagents may afford an accurate means of detecting homophile IgG RF producing cells using other assay systems.

  19. Antibodies to Lactobacilli and Bifidobacteria in young children with different propensity to develop islet autoimmunity.

    Science.gov (United States)

    Talja, Ija; Kubo, Anna-Liisa; Veijola, Riitta; Knip, Mikael; Simell, Olli; Ilonen, Jorma; Vähä-Mäkilä, Mari; Sepp, Epp; Mikelsaar, Marika; Utt, Meeme; Uibo, Raivo

    2014-01-01

    The intestinal microbiota is essential to the maturation and homeostasis of the immune system. Immunoblot assays were used to establish the prevalence of serum IgG, IgM, and IgA antibodies specific for Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus rhamnosus GG proteins in young children presenting with or without type 1 diabetes (T1D). We demonstrated that children between the ages of 6 and 12 months had a substantial increase in the frequency of IgG antibodies specific for L. rhamnosus GG proteins. We measured IgG, IgM, and IgA class antibody reactivity against B. adolescentis DSM 20083, B. adolescentis DSM 20086, and B. longum DSM 20088 proteins demonstrating significantly higher IgA responses against B. adolescentis DSM 20083 strain proteins in children who developed islet autoimmunity and T1D later in life. B. adolescentis strains showed more IgM type antibodies in children who developed T1D later in life, but the difference was not statistically significant. B. longum proteins were recognized by IgG and IgA antibodies to a higher extent compared to other bacteria studied. These results confirm that differences in immune reactivity against some commensal strains in young children may represent a different risk factor for developing T1D.

  20. Avidity of IgG for rubella: an evaluation of the need for implementation at the Materno-Infantil Presidente Vargas Hospital in Porto Alegre, Rio Grande do Sul, Brazil

    Directory of Open Access Journals (Sweden)

    M.M. Reis

    Full Text Available Rubella serum assays performed in the laboratory of the Materno-Infantil Presidente Vargas Hospital (HMIPV from 1998 to 2002 were reviewed to determine if IgG avidity assays should be implemented. IgG was determined using the Enzyme Linked Fluorescent Assay, ELFA, VIDAS® system, bioMérieux or the Microparticle Enzyme Immunoassay, MEIA, Axsym® system, Abbott, and IgM was determined using the ELFA, VIDAS® system, bioMérieux, a capture format assay. Specific IgG was assayed in 2,863 samples, with positive results for 84% of the patients, for the most part with high levels of antibodies. IgM was assayed in 2,851 samples, being positive in 14 (0.49% and inconclusive in 25 (0.88%. Serology for toxoplasmosis was also positive or inconclusive in 5 patients. After a cost-effectiveness analysis, it was decided not to implement avidity assays, considering that the HMIPV is a public institution, with limited funding. Difficulties concerning the integration of the Clinical Pathology Service with the Clinical Staff of the institution were also considered.

  1. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    National Research Council Canada - National Science Library

    Palys, Thomas J; Schmid, Kara E; Scherer, John M; Schoepp, Randal J

    2006-01-01

    Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives...

  2. Serum IgG antibody levels to periodontal microbiota are associated with incident Alzheimer disease.

    Science.gov (United States)

    Noble, James M; Scarmeas, Nikolaos; Celenti, Romanita S; Elkind, Mitchell S V; Wright, Clinton B; Schupf, Nicole; Papapanou, Panos N

    2014-01-01

    Periodontitis and Alzheimer disease (AD) are associated with systemic inflammation. This research studied serum IgG to periodontal microbiota as possible predictors of incident AD. Using a case-cohort study design, 219 subjects (110 incident AD cases and 109 controls without incident cognitive impairment at last follow-up), matched on race-ethnicity, were drawn from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a cohort of longitudinally followed northern Manhattan residents aged >65 years. Mean follow-up was five years (SD 2.6). In baseline sera, serum IgG levels were determined for bacteria known to be positively or negatively associated with periodontitis (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans Y4, Treponema denticola, Campylobacter rectus, Eubacterium nodatum, and Actinomyces naeslundii genospecies-2). In all analyses, we used antibody threshold levels shown to correlate with presence of moderate-severe periodontitis. Mean age was 72 years (SD 6.9) for controls, and 79 years (SD 4.6) for cases (pthe sample. In a model adjusting for baseline age, sex, education, diabetes mellitus, hypertension, smoking, prior history of stroke, and apolipoprotein E genotype, high anti-A. naeslundii titer (>640 ng/ml, present in 10% of subjects) was associated with increased risk of AD (HR = 2.0, 95%CI: 1.1-3.8). This association was stronger after adjusting for other significant titers (HR = 3.1, 95%CI: 1.5-6.4). In this model, high anti-E. nodatum IgG (>1755 ng/ml; 19% of subjects) was associated with lower risk of AD (HR = 0.5, 95%CI: 0.2-0.9). Serum IgG levels to common periodontal microbiota are associated with risk for developing incident AD.

  3. Monovalent IgG4 molecules

    Science.gov (United States)

    Wilkinson, Ian C.; Fowler, Susan B.; Machiesky, LeeAnn; Miller, Kenneth; Hayes, David B.; Adib, Morshed; Her, Cheng; Borrok, M. Jack; Tsui, Ping; Burrell, Matthew; Corkill, Dominic J.; Witt, Susanne; Lowe, David C.; Webster, Carl I.

    2013-01-01

    Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments. PMID:23567207

  4. Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

    Science.gov (United States)

    Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

    2016-10-21

    This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

  5. 125I-Clq-binding and specific antibodies as indicators of pulmonary disease activity in cystic fibrosis

    International Nuclear Information System (INIS)

    Moss, R.B.; Hsu, Y.P.; Lewiston, N.J.

    1981-01-01

    We studied the incidence and levels of circulating immune complexes by the 125 I-Clq-binding assay in patients with cystic fibrosis in relation to clinical respiratory status and specific IgG and IgE antibodies to Pseudomonas aeruginosa. Staphylococcus aureus, Aspergillus fumigatus, and Candida albicans. Overall prevalence of CIC was 43%, but 86% of serially studied patients had evidence of CIC at some time. Patients with acute respiratory exacerbations and deteriorating pulmonary function had a higher incidence of CIC (76%) as compared to stable patients (36%, P less than 0.01), as well as significantly higher levels of CIC. Acute exacerbations were also associated with significant increases in IgG antibody to Pseudomonas (P less than 0.005) but not in other antibodies. CIC did not correlate with Pseudomonas-specific IgG nor with any other specific antibody studied. A variety of age-related differences in specific antibody levels were seen. The episodic appearance of CIC is common in CF and is usually associated with exacerbation of lung disease

  6. Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

    Directory of Open Access Journals (Sweden)

    A.M. Atta

    2008-06-01

    Full Text Available Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and β2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0% hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95%CI: 21.5-25.4 MPL, only three carriers (<3% had IgG anticardiolipin antibodies (median, 23 GPL; 95%CI: 20.5-25.5 GPL. Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004. IgA anti-β2-glycoprotein-I antibodies were detected in 29 of 109 (27.0% hepatitis C carriers (median, 41 SAU; 95%CI: 52.7-103.9 SAU. Twenty patients (18.0% had IgM anti-β2-glycoprotein I antibodies (median, 27.6 SMU; 95%CI: 23.3-70.3 SMU, while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU. Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-β2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.

  7. Antibodies to lactalbumin interfere with its radioimmunoassay in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, U; Laurence, D J.R. [Royal Marsden Hospital, London (UK); Ormerod, M G [Institute of Cancer Research, Sutton (UK). Surrey Branch

    1978-01-01

    Two radioimmunoassays for human lactalbumin have been established using a rabbit antiserum. One assay uses a second antibody to separate bound from free label; the other uses polyethylene glycol to precipitate gamma globulin non-specifically. It is confirmed that about half the normal human population have a substance in their blood which inhibits the binding of lactalbumin to the rabbit antibody. Comparison of the two assays has demonstrated that this material is not lactalbumin but a naturally occurring antibody. It is shown that it is in the IgG fraction of human plasma and is probably a cross-reacting antibody to bovine lactalbumin. None out of fifteen males and fourteen out of fifty eight non-pregnant, non-lacatating females had low levels of lactalbumin in their blood (0.6-2.0 ng/ml). The assay could not detect a statistically significant difference between normal women and those with either benign breast disease or metastatic mammary carcinoma.

  8. IgG4 autoantibodies are inhibitory in the autoimmune disease bullous pemphigoid.

    Science.gov (United States)

    Zuo, Yagang; Evangelista, Flor; Culton, Donna; Guilabert, Antonio; Lin, Lin; Li, Ning; Diaz, Luis; Liu, Zhi

    2016-09-01

    The IgG4 subclass of antibodies exhibits unique characteristics that suggest it may function in an immunoregulatory capacity. The inhibitory function of IgG4 has been well documented in allergic disease by the demonstration of IgG4 blocking antibodies, but similar functions have not been explored in autoimmune disease. Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease characterized by autoantibodies directed against BP180 and an inflammatory infiltrate including eosinophils and neutrophils. Animal models have revealed that the NC16A region within BP180 harbors the critical epitopes necessary for autoantibody mediated disease induction. BP180 NC16A-specific IgG belong to the IgG1, IgG3, and IgG4 subclasses. The purpose of this study was to determine effector functions of different IgG subclasses of NC16A-specific autoantibodies in BP. We find that IgG4 anti-NC16A autoantibodies inhibit the binding of IgG1 and IgG3 autoantibodies to the NC16A region. Moreover, IgG4 anti-NC16A blocks IgG1 and IgG3 induced complement fixation, neutrophil infiltration, and blister formation clinically and histologically in a dose-dependent manner following passive transfer to humanized BP180-NC16A mice. These findings highlight the inhibitory role of IgG4 in autoimmune disease and have important implications for the treatment of BP as well as other antibody mediated inflammatory and autoimmune diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

    Science.gov (United States)

    Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

    2009-08-01

    A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

  10. Antiradiation Antitoxin IgG : Immunological neutralization of Radiation Toxins at Acute Radiation Syndromes.

    Science.gov (United States)

    Popov, Dmitri; Maliev, Slava

    radiation toxins to induce hyperimmune serum: Group A -Toxoid form of CV ARS toxins ( SRD-1); Group B-Toxoid form of CR ARS (SRD-2)toxins ; Group C -Toxoid form of GI ARS (SRD-3); Group D -Toxoid form of HP ARS (SRD-4). After the hyperimmune serum was pooled from several animals, purified, and concentrated, the IgG fraction was separated. Enzyme-linked immunosorbent assays of the hyper-immune serum had revealed high titers of IgG with specific binding to radi-ation toxins. The antiradiation IgG preparation was injected into laboratory animals one hour before and three hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. Results: Animals that were inoculated with specific antiradiation antibodies before and after receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate within 30 days. Also, these animals inoculated with the Antiradiation Antitoxin had exhibited markedly reduced clinical symptoms of the ARS, even those ones that did not survive irradiation. Discussion: The results of our experiments have demonstrated that the rabbit hyperimmune IgG preparations directed against SRD toxins provide a significant protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of animals treated by the hyperimmune IgG antidote have varied in the different groups of ani-mals and different forms of the ARS. However, significant radioprotection was observed in each group treated with the IgGs. The specific antiradiation antidote IGg isolated from hyperim-mune serum of immunized horses is under study. The specific antiradiation antidote contains antibodies to neurotoxins -SAAN IgG includes 50% IgG to Cv ARS, 25% IgG to Cr ARS and 25 % IgG to Gi ARS. The other type of the Specific antiradiation antidote containes antibodies to hematotoxins -SAAH IgG -100%. A combined variant is under

  11. The prevalence of toxoplasma igG and IgM in pregnant women residing in Rawalpindi

    International Nuclear Information System (INIS)

    Kausar, N.; Akhtar, S.; Ikram, A.

    2010-01-01

    Objective: To determine the prevalence of Toxoplasma gondii antibodies (IgG/IgM) among pregnant women visiting Military Hospital Rawalpindi and to develop a relationship between various risk factors and disease prevalence. Methods: One thousand pregnant women reporting in out patient Gynaecology department of Military Hospital (MH) Rawalpindi from October 2008 through January 2009 for antenatal check up were included in the study. Their serum samples were tested for the presence of Toxoplasma IgM and IgG immunoglobulins. Enzyme Linked immunosorbent assay test kits for both IgG and IgM were used to detect 1: gondii immunoglobulins in serum samples. Rest of the serum was stored at -20 degree C. Results: Of the 1000 women sampled at hospital, 46 (4.6%) had evidence of past infection and were seropositive for immunoglobulins of T. gondii IgG, while none of them were seropositive for IgM immunoglobulin, suggesting absence of recent infections during pregnancy. Conclusion: In twin cities of Islamabad and Rawalpindi, the sero prevalence of T. gondii IgG in pregnant women is relatively high (4.6%) as compared to other areas nearby. Consequently, the risk of, primary infection during pregnancy and the potential for congenital infection of foetus remains there as a large number of pregnant women were sero-negative for both the antibodies.

  12. The role of IgG antibodies in allergy and immunotherapy

    NARCIS (Netherlands)

    Aalberse, R.

    2011-01-01

    In specific immunotherapy (SIT), a beneficial response is associated with an increase in allergen-specific IgG(4) . This does not indicate that IgE-producing B cells have switched to IgG(4) production, because in human DNA, IgE is downstream from IgG(4) . Thus, by conventional switching, B cells

  13. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  14. Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

    Science.gov (United States)

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-01-01

    The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease. PMID:26137589

  15. Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination

    Directory of Open Access Journals (Sweden)

    Sven Kratochvil

    2017-12-01

    Full Text Available Antibody subclasses exhibit extensive polymorphisms (allotypes that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1 of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC and antibody-dependent cellular phagocytosis (ADCP function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

  16. Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

    Science.gov (United States)

    Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

    2017-09-01

    Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  17. Genetic and infectious profiles influence cerebrospinal fluid IgG abnormality in Japanese multiple sclerosis patients.

    Directory of Open Access Journals (Sweden)

    Satoshi Yoshimura

    Full Text Available BACKGROUND: Abnormal intrathecal synthesis of IgG, reflected by cerebrospinal fluid (CSF oligoclonal IgG bands (OBs and increased IgG index, is much less frequently observed in Japanese multiple sclerosis (MS cohorts compared with Western cohorts. We aimed to clarify whether genetic and common infectious backgrounds influence CSF IgG abnormality in Japanese MS patients. METHODOLOGY: We analyzed HLA-DRB1 alleles, and IgG antibodies against Chlamydia pneumoniae, Helicobacter pylori, Epstein-Barr virus nuclear antigen (EBNA, and varicella zoster virus (VZV in 94 patients with MS and 367 unrelated healthy controls (HCs. We defined CSF IgG abnormality as the presence of CSF OBs and/or increased IgG index (>0.658. PRINCIPAL FINDINGS: CSF IgG abnormality was found in 59 of 94 (62.8% MS patients. CSF IgG abnormality-positive patients had a significantly higher frequency of brain MRI lesions meeting the Barkhof criteria compared with abnormality-negative patients. Compared with HCs, CSF IgG abnormality-positive MS patients showed a significantly higher frequency of DRB1 1501, whereas CSF IgG abnormality-negative patients had a significantly higher frequency of DRB1 0405. CSF IgG abnormality-positive MS patients had a significantly higher frequency of anti-C. pneumoniae IgG antibodies compared with CSF IgG abnormality-negative MS patients, although there was no difference in the frequency of anti-C. pneumoniae IgG antibodies between HCs and total MS patients. Compared with HCs, anti-H. pylori IgG antibodies were detected significantly less frequently in the total MS patients, especially in CSF IgG abnormality-negative MS patients. The frequencies of antibodies against EBNA and VZV did not differ significantly among the groups. CONCLUSIONS: CSF IgG abnormality is associated with Western MS-like brain MRI features. DRB1 1501 and C. pneumoniae infection confer CSF IgG abnormality, while DRB1 0405 and H. pylori infection are positively and negatively

  18. Seroprevalence of toxoplasma-specific antibodies in patients suspected to have active toxoplasmosis: A cross-sectional survey

    Directory of Open Access Journals (Sweden)

    Abbas Ali Eskandarian

    2014-01-01

    Full Text Available Background: The aim of this study was to investigate the presence and distribution of anti-toxoplasma-specific IgM and IgG tantibodies in patients suspected to have toxoplasmosis and investigate for any association between IgM and IgG antibodies and some toxoplasmosis risk factors as well. Materials and Methods: In a comparative cross-sectional study, 70 patients suspected to had active toxoplasmosis and 30 control volunteers, who gave informed consent, entered the study. In each group, patient age, sex, signs of appearance, education level, residency status (urban / rural, occupation, frequency of toxoplasma-specific IgG and IgM antibodies, abortion history, and some risk factors (Direct cat exposure, Occupational exposure to raw meat, and Raw vegetable consumption were recorded. The enzyme-linked immunosorbent assay (ELISA kits (EUROIMMUN®, United Kingdom were used for the evaluation of anti-toxoplasma IgG and IgM antibodies according to the manufacturer›s instructions. All analyses were done using SPSS-20. Results: The frequency of toxoplasma-specific IgG and IgM antibodies like: Direct cat exposures, Occupational exposure to raw meat, and Raw vegetable consumption were not statistically significant between the two groups (P > 0.05. The history of previous abortions in women in the toxoplasmosis-suspected group was significantly higher than that in the controls (31.4% versus 6.7%; P = 0.009. Conclusion: The frequency of specific IgM and IgG antibodies in toxoplasmosis suspected in the toxoplasmosis and control groups was not statistically significant.

  19. Ventricular assist device elicits serum natural IgG that correlates with the development of primary graft dysfunction following heart transplantation.

    Science.gov (United States)

    See, Sarah B; Clerkin, Kevin J; Kennel, Peter J; Zhang, Feifan; Weber, Matthew P; Rogers, Kortney J; Chatterjee, Debanjana; Vasilescu, Elena R; Vlad, George; Naka, Yoshifumi; Restaino, Susan W; Farr, Maryjane A; Topkara, Veli K; Colombo, Paolo C; Mancini, Donna M; Schulze, P Christian; Levin, Bruce; Zorn, Emmanuel

    2017-08-01

    Pre-transplant sensitization is a limiting factor in solid-organ transplantation. In heart transplants, ventricular assist device (VAD) implantation has been associated with sensitization to human leukocyte antigens (HLA). The effect of VAD on non-HLA antibodies is unclear. We have previously shown that polyreactive natural antibodies (Nabs) contribute to pre-sensitization in kidney allograft recipients. Here we assessed generation of Nabs after VAD implantation in pre-transplant sera and examined their contribution to cardiac allograft outcome. IgM and IgG Nabs were tested in pre-transplant serum samples collected from 206 orthotopic heart transplant recipients, including 128 patients with VAD (VAD patients) and 78 patients without VAD (no-VAD patients). Nabs were assessed by testing serum reactivity to apoptotic cells by flow cytometry and to the generic oxidized epitope, malondialdehyde, by enzyme-linked immunosorbent assay. No difference was observed in serum levels of IgM Nabs between VAD and no-VAD patients. However, serum IgG Nabs levels were significantly increased in VAD compared with no-VAD patients. This increase was likely due to the presence of the VAD, as revealed by lower serum IgG Nabs levels before implantation. Elevated pre-transplant IgG Nabs level was associated with development of primary graft dysfunction (PGD). Our study demonstrates that VAD support elicits IgG Nabs reactive to apoptotic cells and oxidized epitopes. These findings further support broad and non-specific B-cell activation by VAD, resulting in IgG sensitization. Moreover, the association of serum IgG Nabs levels with development of PGD suggests a possible role for these antibodies in the inflammatory reaction accompanying this complication. Copyright © 2017 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  20. Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

    Science.gov (United States)

    Pardridge, William M

    2015-02-01

    Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

  1. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John

    2008-01-01

    -based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...... reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma....... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1...

  2. Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Bergmann, S M; Wang, Q; Zeng, W; Li, Y; Wang, Y; Matras, M; Reichert, M; Fichtner, D; Lenk, M; Morin, T; Olesen, N J; Skall, H F; Lee, P-Y; Zheng, S; Monaghan, S; Reiche, S; Fuchs, W; Kotler, M; Way, K; Bräuer, G; Böttcher, K; Kappe, A; Kielpinska, J

    2017-11-01

    Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs. © 2017 John Wiley & Sons Ltd.

  3. STUDY ON SEROPREVALENCE OF MUMPS - SPECIFIC IgG ANTIBODIES IN A HEALTHY POPULATION

    Directory of Open Access Journals (Sweden)

    Milena Karcheva

    2010-09-01

    Full Text Available Mumps is a vaccine preventable viral infection. Its typical clinical manifestations are characterized by pain and swelling of the salivary glands, fever, and fatigue. Often other organs are affected - testes in males after puberty (orchitis, ovaries in women (ooforitis, pancreas (pancreatitis, central nervous system (meningities. The use of specific immune prophylaxis led to a significant success in the fight against mumps, but there are still unresolved issues related to the immunological and epidemiological effectiveness of the vaccines. The disease continues to interest researchers today. The main issues being tackled are related to the conduct of virological, clinical and sero-epidemiological studies in different countries. Objectives of the study is to determine the frequency distribution of mumps-specific IgG antibodies in healthy populations in the region of Pleven, Bulgaria. Methods: a cross-sectional sero - epidemiological representative population - based survey in the area was made. Enzyme immunoassay method was used for an indirect proof of mumps - specific IgG serum antibodies. 410 people were examined at an average age of 25 (1 to 84. Of these, 250 (61 % were women and 160 (39 % - men. Results: Of all test results, the negative were 72 (19 %, the borderline were 12 (3 %, the positive were 182 (44 %, and highly positive were 144 (35 %. The vaccination status showed that 242 (69 % of all surveyed were immunized with a vaccine against mumps. According to the immunization schedule in Bulgaria, 132 (33 % people were immunized with monovaccine during the years - 1 intake, 80 (20 % with trivaccine - 1 intake, and 64 (16 % - 2 doses. Conclusion: We believe that despite the specific immunprophylaxis carried out against mumps decades on end, the necessary level of protection leading to its elimination has not yet been reached.

  4. A simple assay for the detection of antibodies to endocrine islet cell surface antigens

    International Nuclear Information System (INIS)

    Contreas, G.; Madsen, O.D.; Vissing, H.; Lernmark, Aa.

    1986-01-01

    A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125 I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens. (Auth.)

  5. IgG immune responses to different proteins of Helicobacter Pylori as defined by immunoblot assay

    Directory of Open Access Journals (Sweden)

    Raeiszadeh M

    2000-09-01

    Full Text Available Helicobacter pylori (H.pylori is an etiologic factor for chronic gastritis and peptic ulcers. Serological testing of H.pylori infection is common in Iran, as other parts of the world. There are geographical variations in the humoral immune response to various H. pylori strains in different parts of the worl. We studied the immunogenic proteins of H.pylori by means of an Immunoblot assay with antigens of H.pylori strains isolated in Iran. Sera of 64 patients suffering from dyspepsia were analyzed to determine antibodlies which were good marker of infection and the antibody patterns associated with peptic ulcer.54 out of 64 dyspeptic patients were infected by H. pylori based on positive culture or positive results of both rapid urease test and direct examination. 14 out of fity-four had peptic ulcers and the rest were catagoriied as patients with non-ulcer dyspepsia. Some of them had multiple erosions in the gut or deodenum. Tweny –two major bands were identified by immunoblot. Of these, IgG antibodies against 10 protients, and they produced immunoreative bands at 14, 16, 22, 26, 32 , 32, 44, 87, 92, 120 Kda. Antibody patterns were not identical in the patients. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection(sensitivity, 90% and specificity, 80% Major serological cross reactions were found at moderate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection (sensitivity, 90% and specificity, 80%. Major serological crossreactions were found at moderate molerate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of antibodies to 120 Kda protein (Cag A and 87 Kda Protein (Vac A were not associated with the presence of peptic ulcers. These were in contradiction to results obtained across Europe and U.S but in agreement with Asian studies. However the presence of at least one band at either 32 or 35 Kda was

  6. An electric detection of immunoglobulin G in the enzyme-linked immunosorbent assay using an indium oxide nanoparticle ion-sensitive field-effect transistor

    International Nuclear Information System (INIS)

    Lee, Dongjin; Cui, Tianhong

    2012-01-01

    Semiconducting nanoparticle ion-sensitive field-effect transistors (ISFETs) are used to detect immunoglobulin G (IgG) in the conventional enzyme-linked immunosorbent assay (ELISA). Indium oxide and silica nanoparticles were layer-by-layer self-assembled with the oppositely charged polyelectrolyte as the electrochemical transducer and antibody immobilization site, respectively. The assay was conducted on a novel platform of indium oxide nanoparticle ISFETs, where the electric signals are generated in response to the concentration of target IgG using the labeled detecting antibody. The sandwiched ELISA structure catalyzed the conversion of the acidic substrate into neutral substance with the aid of horseradish peroxidase. The pH change in the substrate solution was detected by nanoparticle ISFETs. Normal rabbit IgG was used as a model antigen whose detection limit of 0.04 ng ml −1 was found. The facile electric detection in the conventional assay through the semiconducting nanoparticle ISFET has potential applications as a point-of-care detection or a sensing element in a lab-on-a-chip system

  7. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

    Science.gov (United States)

    2016-02-01

    Enzyme- linked immunosorbent assay (ELISA) Quality Testing MS2 coat protein (MS2CP) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...primarily relied on the performance of an antibody in an enzyme- linked immunosorbent assay (ELISA), with little regard for quantifying the full spectrum...Protection, and Multi-Year Stabilization, in High Concentration Protein Solutions, Using Ionic Liquids. Chem. Commun. (Camb) 2007, 26, 2714–2716. 3. Bio

  8. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

    Directory of Open Access Journals (Sweden)

    Amy E Gilbert

    Full Text Available Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10 to primary and metastatic melanoma cells compared to healthy volunteers (n = 10 (P<0.0001. Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21 (P<0.0001. Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800 compared to 2% of cultures from healthy controls (n = 600 produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  9. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    Science.gov (United States)

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  10. Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

    NARCIS (Netherlands)

    Irmak, Hasan; Buzgan, Turan; Evirgen, Omer; Akdeniz, Hayrettin; Demiroz, A. Pekcan; Abdoel, Theresia H.; Smits, Henk L.

    2004-01-01

    The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in

  11. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  12. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  13. Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG

    International Nuclear Information System (INIS)

    Carson, D.A.; Lawrance, S.; Catalano, M.A.; Vaughan, J.H.; Abraham, G.

    1977-01-01

    Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation

  14. A rare case of Addison's disease, hepatitis, thyreoiditis, positive IgG anti-tissue transglutaminase antibodies and partial IgA deficiency.

    Science.gov (United States)

    Baleva, Marta P; Mihaylova, Snejina; Yankova, Petja; Atanasova, Iliana; Nikolova-Vlahova, Milena; Naumova, Elissaveta

    2016-01-01

    Selective IgA deficiency (IgAD) is the most prevalent type of primary immune deficiencies, but partial IgA deficiency is even more common. Addison's disease is a rare condition associated with primary adrenal insufficiency due to infection or autoimmune destruction of the adrenals. The association between IgA deficiency and Addison's disease is very rare. We observed a 22-year-old male patient with marked darkening of the skin, especially on the palms and areolae, jaundice on the skin and sclera, astheno-adynamia, hypotension (80/50 mm Hg), and pain in the right hypochondrium. The laboratory investigations revealed increased serum levels of total and indirect bilirubin, AST, ALT, GGT and LDH, negative HBsAg, anti-HBc IgM, anti-HCV and anti-HAV IgM, very low serum IgA levels (0.16 g/l) with normal IgG and IgM, negative ANA, ANCA, AMA, LKM-1, anti-GAD-60, anti-IA-2, anti-thyroglobulin antibodies, a mild increase in anti-TPO antibodies titer, a marked increase in IgG anti-tissue transglutaminase antibodies, with no typical changes in cellular immunity, negative T-SPOT-TB test, HLA - A*01; B*08; DRB1*03; DQB1*02, karyotype - 46, XY. We present a rare case of partial IgA deficiency with Addison's disease, hepatitis, thyroiditis and positive anti-tissue transglutaminase antibodies. IgAD and some autoimmune disorders share several predisposing HLA genes, thus explaining the increased prevalence of IgAD in certain patient groups.

  15. Thrombosis and antiphospholipid antibody syndrome during acute Q fever: A cross-sectional study.

    Science.gov (United States)

    Million, Matthieu; Bardin, Nathalie; Bessis, Simon; Nouiakh, Nadia; Douliery, Charlaine; Edouard, Sophie; Angelakis, Emmanouil; Bosseray, Annick; Epaulard, Olivier; Branger, Stéphanie; Chaudier, Bernard; Blanc-Laserre, Karine; Ferreira-Maldent, Nicole; Demonchy, Elisa; Roblot, France; Reynes, Jacques; Djossou, Felix; Protopopescu, Camelia; Carrieri, Patrizia; Camoin-Jau, Laurence; Mege, Jean-Louis; Raoult, Didier

    2017-07-01

    Q fever is a neglected and potentially fatal disease. During acute Q fever, antiphospholipid antibodies are very prevalent and have been associated with fever, thrombocytopenia, acquired heart valve disease, and progression to chronic endocarditis. However, thrombosis, the main clinical criterion of the 2006 updated classification of the antiphospholipid syndrome, has not been assessed in this context. To test whether thrombosis is associated with antiphospholipid antibodies and whether the criteria for antiphospholipid syndrome can be met in patients with acute Q fever, we conducted a cross-sectional study at the French National Referral Center for Q fever.Patients included were diagnosed with acute Q fever in our Center between January 2007 and December 2015. Each patient's history and clinical characteristics were recorded with a standardized questionnaire. Predictive factors associated with thrombosis were assessed using a rare events logistic regression model. IgG anticardiolipin antibodies (IgG aCL) assessed by an enzyme-linked immunosorbent assay were tested on the Q fever diagnostic serum. A dose-dependent relationship between IgG aCL levels and thrombosis was tested using a receiver operating characteristic (ROC) analysis.Of the 664 patients identified for inclusion in the study, 313 (47.1%) had positive IgG aCL and 13 (1.9%) were diagnosed with thrombosis. Three patients fulfilled the antiphospholipid syndrome criteria. After multiple adjustments, only positive IgG aCL (relative risk, 14.46 [1.85-113.14], P = .011) were independently associated with thrombosis. ROC analysis identified a dose-dependent relationship between IgG aCL levels and occurrence of thrombosis (area under curve, 0.83, 95%CI [0.73-0.93], P antiphospholipid antibodies are associated with thrombosis, thrombocytopenia, and acquired valvular heart disease. Antiphospholipid antibodies should be systematically assessed in acute Q fever patients. Hydroxychloroquine, which has been

  16. Comparison of semi-automatized assays for anti-T. gondii IgG detection in low-reactivity serum samples: importance of the results in patient counseling Comparação de ensaios semi-automatizados para pesquisa de IgG anti-T. gondii em amostras de soros de baixa reatividade: importância dos resultados no aconselhamento do paciente

    Directory of Open Access Journals (Sweden)

    Paulo Guilherme Leser

    2003-06-01

    Full Text Available Toxoplasmosis is a disease which can cause severe congenital infection and is normally diagnosed by the detection of T. gondii specific antibodies in the serum of infected patients. Several different tests allow to distinguish recent from past infections and to quantify anti-T. gondii specific IgG, and the results can be used as markers for immunity. In the present study, we compare the performance of two different methodologies, the Elfa (bioMérieux S.A and the Meia (Abbott Laboratories in detecting T. gondii specific IgG in low-reactivity sera. Of 76 analyzed samples, three presented discrepant results, being positive in the Abbott AxSYM Toxo IgG assay, and negative in the bioMérieux Vidas Toxo IgG II assay. By using other tests, the three sera were confirmed to be negative. The results are discussed in the context of their importance for patient management, especially during pregnancy.Toxoplasmose, doença conhecida por sua severidade na infecção congênita é geralmente diagnosticada pela demonstração de anticorpos específicos contra antígenos de T. gondii, presentes no soro de indivíduos infectados. Diferentes testes são disponíveis para diferenciar infecção recente de infecção pregressa, para quantificar anticorpos IgG anti-T. gondii nos soros dos pacientes e utilizar os resultados como marcadores de imunidade. Neste trabalho apresentamos os resultados do estudo comparativo de duas tecnologias, Elfa (bioMérieux S.A. e Meia (Abbott Laboratories, para pesquisa de anticorpos IgG anti-T. gondii em amostras de soros de baixa reatividade. De 76 amostras processadas, três apresentaram resultados discrepantes, reagentes para AxSYM Toxo IgG e não-reagentes para Vidas Toxo IgG II. A confirmação dos resultados, feita por bateria de testes, mostrou que todas as três amostras eram não-reagentes. Os resultados são discutidos em sua importância e orientação clínica, principalmente para a paciente gestante.

  17. IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

    Science.gov (United States)

    Groh, N; von Loetzen, C S; Subbarayal, B; Möbs, C; Vogel, L; Hoffmann, A; Fötisch, K; Koutsouridou, A; Randow, S; Völker, E; Seutter von Loetzen, A; Rösch, P; Vieths, S; Pfützner, W; Bohle, B; Schiller, D

    2017-05-01

    Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G 4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG 4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG 4 antibodies are developed is under debate. We sought to analyze the epitope specificities of IgE and IgG 4 antibodies from sera of patients who received AIT. 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG 4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a _11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG 4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. rBet v 1a _11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a _11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC 50 for IgE-mediated mediator release induced by rBet v 1a _11x was determined. The reduction of IgE and IgG 4 binding to rBet v 1a _11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG 4 to 66.1% and 64.9%, respectively. Serum IgE and IgG 4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. Patients receiving AIT develop Bet v 1a-specific IgG 4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG 4 might stimulate the development of epitope-specific diagnostics and therapeutics. © 2016 John Wiley & Sons Ltd.

  18. Total lymphoid irradiation reduces IgG autoantibody production and enhances specific antibody responses in NZB/NZW F1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Farinas, M.C.; Strober, S.

    1989-07-01

    Thymus-independent primary antibody responses were studied in young and old (9 months) untreated and TLI-treated NZB/NZW and BALB/c mice. Untreated old NZB/NZW mice had a low primary response to Brucella abortus (BA) as compared to that of young NZB/NZW and BALB/c mice. However, TLI treatment resulted in a 130-fold increase in the IgG anti-BA primary antibody response at day 21 postimmunization, achieving similar levels to those of young NZB/NZW or nonautoimmune BALB/c mice. Anti-TNP responses to trinitrophenylated BA or Ficoll were masked by high background levels of anti-TNP antibodies. Despite the increase in the anti-BA response, spontaneous immunoglobulin secretion and autoantibody levels were markedly decreased after TLI in old NZB/NZW mice.

  19. Total lymphoid irradiation reduces IgG autoantibody production and enhances specific antibody responses in NZB/NZW F1 mice

    International Nuclear Information System (INIS)

    Farinas, M.C.; Strober, S.

    1989-01-01

    Thymus-independent primary antibody responses were studied in young and old (9 months) untreated and TLI-treated NZB/NZW and BALB/c mice. Untreated old NZB/NZW mice had a low primary response to Brucella abortus (BA) as compared to that of young NZB/NZW and BALB/c mice. However, TLI treatment resulted in a 130-fold increase in the IgG anti-BA primary antibody response at day 21 postimmunization, achieving similar levels to those of young NZB/NZW or nonautoimmune BALB/c mice. Anti-TNP responses to trinitrophenylated BA or Ficoll were masked by high background levels of anti-TNP antibodies. Despite the increase in the anti-BA response, spontaneous immunoglobulin secretion and autoantibody levels were markedly decreased after TLI in old NZB/NZW mice

  20. Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

    International Nuclear Information System (INIS)

    Kasprzyk, P.G.; Cuttitta, F.; Avis, I.; Nakanishi, Y.; Treston, A.; Wong, H.; Walsh, J.H.; Mulshine, J.L.

    1988-01-01

    A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones

  1. Incorporation of FcRn-mediated disposition model to describe the population pharmacokinetics of therapeutic monoclonal IgG antibody in clinical patients.

    Science.gov (United States)

    Ng, Chee M

    2016-03-01

    The two-compartment linear model used to describe the population pharmacokinetics (PK) of many therapeutic monoclonal antibodies (TMAbs) offered little biological insight to antibody disposition in humans. The purpose of this study is to develop a semi-mechanistic FcRn-mediated IgG disposition model to describe the population PK of TMAbs in clinical patients. A standard two-compartment linear PK model from a previously published population PK model of pertuzumab was used to simulate intensive PK data of 100 subjects for model development. Two different semi-mechanistic FcRn-mediated IgG disposition models were developed and First Order Conditional Estimation (FOCE) with the interaction method in NONMEM was used to obtain the final model estimates. The performances of these models were then compared with the two-compartment linear PK model used to simulate the data for model development. A semi-mechanistic FcRn-mediated IgG disposition model consisting of a peripheral tissue compartment and FcRn-containing endosomes in the central compartment best describes the simulated pertuzumab population PK data. This developed semi-mechanistic population PK model had the same number of model parameters, produced very similar concentration-time profiles but provided additional biological insight to the FcRn-mediated IgG disposition in human subjects compared with the standard linear two-compartment linear PK model. This first reported semi-mechanistic model may serve as an important model framework for developing future population PK models of TMAbs in clinical patients. Copyright © 2015 John Wiley & Sons, Ltd.

  2. NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

    Directory of Open Access Journals (Sweden)

    Yoshie Kametani

    Full Text Available Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD, which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg. After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2 cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP or keyhole limpet hemocyanin (KLH successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

  3. Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia; Svenson, Morten

    2009-01-01

    activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution...

  4. Correlation of Fc(gamma)RIIa (CD32) Polymorphism and IgG Antibody Subclasses in Hemolytic Disease of Newborn.

    Science.gov (United States)

    Wu, QiangJu; Zhang, Yan; Liu, MengLi; Wang, Bo; Liu, Sheng; He, Chen

    2009-01-01

    ABO-HDN is a common disease of newborn in China and currently there is no satisfactory method to predict it in the antepartum period. It has been reported that Fc(gamma)RIIa (CD32) genotype is associated with both infectious diseases induced by bacteria and parasitemia. There is a relationship between IgG subclass and RH-HDN. To study the pathogenesis of ABO-HDN and to find reliable method to diagnose ABO-HDN, we investigated the polymorphism of Fc(gamma)RIIa (CD32) and distribution of IgG subclass in infants with ABO-HDN and their mothers by polymerase chain reaction or ELISA assay. We observed that the frequency of HH131 genotype is lower in infants with ABO-HDN than in controls (p < 0.01), while the frequency of HR131 genotype is higher in ABO-HDN infants than that in controls (p < 0.01). The genotype HR131 and concentrations of IgG1 and IgG3 are significantly correlated with ABO-HDN. Our results demonstrated, for the first time, that there is a correlation between ABO-HDN and CD32, and different IgG subclass distribution. Our study may contribute to the development of an early diagnostic method for HDN. Copyright 2009 S. Karger AG, Basel.

  5. Hinge-deleted IgG4 blocker therapy for acetylcholine receptor myasthenia gravis in rhesus monkeys.

    Science.gov (United States)

    Losen, Mario; Labrijn, Aran F; van Kranen-Mastenbroek, Vivianne H; Janmaat, Maarten L; Haanstra, Krista G; Beurskens, Frank J; Vink, Tom; Jonker, Margreet; 't Hart, Bert A; Mané-Damas, Marina; Molenaar, Peter C; Martinez-Martinez, Pilar; van der Esch, Eline; Schuurman, Janine; de Baets, Marc H; Parren, Paul W H I

    2017-04-20

    Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

  6. An Enhanced Pre- and Postnatal Development Study in Cynomolgus Monkeys with Tabalumab: A Human IgG4 Monoclonal Antibody.

    Science.gov (United States)

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Newcomb, Deanna L; Chellman, Gary J

    2015-06-01

    Tabalumab, a human IgG4 monoclonal antibody (mAb) with neutralizing activity against both soluble and membrane B-cell activating factor (BAFF), has been under development for the treatment of autoimmune diseases. The purpose of this study was to determine the potential adverse effects of maternal tabalumab exposure on pregnancy, parturition, and lactation of the mothers and on the growth, viability, and development of the offspring through postnatal day (PND) 204. Tabalumab was administered by subcutaneous injection to presumed pregnant cynomolgus monkeys (16-19 per group) every 2 weeks from gestation day (GD) 20 to 22 until parturition at doses of 0, 0.3, or 30 mg/kg. Evaluations in mothers and infants included clinical signs, body weight, toxicokinetics, blood lymphocyte phenotyping, T-cell-dependent antibody response (infants only), antitherapeutic antibody (ATA), organ weights (infants only), and gross and microscopic histopathology. Infants were also examined for external and visceral morphologic and neurobehavioral development. There were no adverse tabalumab-related effects on maternal or infant endpoints. An expected pharmacological decrease in peripheral blood B-lymphocytes occurred in adults and infants; however, B-cell recovery was evident by PND154 in adults and infants at 0.3 mg/kg and by PND204 in infants at 30 mg/kg. At 30 mg/kg, a reduced IgM antibody response to T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was observed following primary immunization. Following secondary KLH immunization, all infants in both dose groups mounted anti-KLH IgM and IgG antibody responses similar to control. Placental and mammary transfer of tabalumab was demonstrated. In conclusion, the no-observed-adverse-effect level for maternal and developmental toxicity was 30 mg/kg, the highest dose tested. Exposures at 30 mg/kg provide a margin of safety of 16× the anticipated clinical exposure. © 2015 Wiley Periodicals, Inc.

  7. A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

    Directory of Open Access Journals (Sweden)

    Helene Andersson-Svahn

    2011-11-01

    Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  8. Neuromyelitis optica spectrum disorders: comparison of clinical and magnetic resonance imaging characteristics of AQP4-IgG versus MOG-IgG seropositive cases in the Netherlands.

    Science.gov (United States)

    van Pelt, E D; Wong, Y Y M; Ketelslegers, I A; Hamann, D; Hintzen, R Q

    2016-03-01

    Neuromyelitis optica spectrum disorders (NMOSDs) are a group of rare inflammatory demyelinating disorders of the central nervous system. The identification of specific antibodies directed to aquaporin 4 (AQP4-IgG) led to the distinction from multiple sclerosis. However, up to 25% of the clinically diagnosed NMO patients are seronegative for AQP4-IgG. A subgroup of these patients might be identified by antibodies directed to myelin oligodendrocyte glycoprotein (MOG-IgG). Our objective was to investigate whether the clinical characteristics of these patients differ. Using a cell-based assay, samples of 61 AQP4-IgG seronegative patients and 41 AQP4-IgG seropositive patients with clinically NMOSD were analysed for the presence of MOG-IgG. Clinical characteristics of the AQP4-IgG, MOG-IgG seropositive and double seronegative NMOSD patients were compared. Twenty of the 61 AQP4-IgG seronegative patients tested MOG-IgG seropositive (33%). MOG-IgG seropositive patients were more frequently males in contrast to AQP4-IgG seropositive patients (55% vs. 15%, P < 0.01) and Caucasians (90% vs. 63%, P = 0.03). They more frequently presented with coincident optic neuritis and transverse myelitis (40% vs. 12%, P = 0.02) and had a monophasic disease course (70% vs. 29%, P < 0.01). AQP4-IgG seropositive patients were 2.4 times more likely to suffer from relapses compared with MOG-IgG seropositive patients (relative risk 2.4, 95% confidence interval 1.2-4.7). AQP4-IgG seropositive patients had higher Expanded Disability Status Scale levels at last follow-up (P < 0.01). Antibodies directed to MOG identify a subgroup of AQP4-IgG seronegative NMO patients with generally a favourable monophasic disease course. © 2015 EAN.

  9. Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts.

    Science.gov (United States)

    Heskett, Katherine A; Mackay, Robert J

    2008-03-01

    To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. 18 adult horses. 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.

  10. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa

    DEFF Research Database (Denmark)

    Hansen, Johanne Damgaard; Meyrowitsch, Dan W.; Rwegoshora, Rwehumbiza T.

    2016-01-01

    to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4......A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection...

  11. Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

    Directory of Open Access Journals (Sweden)

    Francisco J Quintana

    Full Text Available B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

  12. Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes.

    Science.gov (United States)

    Heyworth, M F

    1992-02-01

    The aim of this work was to examine the ability of mouse IgA, IgG, and IgM anti-Giardia antibodies to kill Giardia muris trophozoites in the presence and absence of complement. Using a 2-color flow cytometry assay, binding of antibody to trophozoites was assessed with fluorescein-conjugated anti-mouse immunoglobulin, and percentages of killed trophozoites were quantified by staining with propidium iodide. Trophozoites were killed in the presence of complement by IgG3 and IgM anti-trophozoite monoclonal antibodies. Anti-trophozoite IgA, obtained from the intestinal lumen of G. muris-infected BALB/c mice, became bound to trophozoites in vitro but did not kill these organisms in the presence or absence of complement. The results suggest that clearance of G. muris infection by intestinal IgA directed against G. muris trophozoites does not involve antibody-dependent killing of trophozoites in the intestinal lumen.

  13. Quantitative and temporal analyses of murine antibody response in serum and gut secretions to infection with Giardia muris.

    Science.gov (United States)

    Snider, D P; Underdown, B J

    1986-04-01

    We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA), IgG, and IgM isotypes, at weekly intervals, over the course of a 7-week infection in BALB/c and C57BL/6 mice. Using sensitive immunoradiometric assays, we observed that IgA antibody was the only detectable anti-G. muris antibody in intestinal secretions throughout the course of infection. No secreted IgG or IgM anti-G. muris antibody was detected even in concentrated intestinal secretions. The expulsion of G. muris by the mice was associated closely with the appearance and increasing levels of secreted anti-G. muris IgA antibody. Both IgG and IgA serum antibody to G. muris were detected, but no serum IgM antibody was detected. Serum IgA and IgG anti-G. muris antibody remained at high levels up to 10 weeks following clearance of the parasite. An interesting observation indicated that serum IgA antibody to G. muris developed more slowly in response to infection than secreted IgA antibody. An analysis of the molecular weight distribution of total serum IgA in infected mice determined that infection produced a transient but significant shift in serum IgA to high-molecular-weight (greater than or equal to dimeric IgA) forms. The results indicate that a substantial IgA antibody response occurs in sera and in gut secretions of G. muris-resistant mice and that IgA antibody is the dominant and possibly the only effector antibody active in intestinal secretions during G. muris infection in mice.

  14. Diagnostic and prognostic value of factor VIII binding antibodies in acquired hemophilia A: data from the GTH-AH 01/2010 study.

    Science.gov (United States)

    Werwitzke, S; Geisen, U; Nowak-Göttl, U; Eichler, H; Stephan, B; Scholz, U; Holstein, K; Klamroth, R; Knöbl, P; Huth-Kühne, A; Bomke, B; Tiede, A

    2016-05-01

    Essentials Factor VIII (FVIII) binding IgG detected by ELISA could be an alternative to the Bethesda assay. We studied the performance of anti-FVIII IgG ELISA in patients with acquired hemophilia and controls. Anti-FVIII IgG > 99th percentile of controls was highly sensitive and specific. Patients with high anti-FVIII IgG have a lower chance of achieving remission. Background Acquired hemophilia A is a severe bleeding disorder that requires fast and accurate diagnosis as it occurs often unexpectedly in previously healthy men and women of every age. The Nijmegen-modified Bethesda assay is the diagnostic reference standard for detecting neutralizing autoantibodies against factor VIII (FVIII), but is not widely available, not ideal for quantifying the complex type 2 inhibitors seen in acquired hemophilia, and suffers from high inter-laboratory variability. Objectives To assess the diagnostic and prognostic value of FVIII-binding antibodies as detected by ELISA compared with the Nijmegen Bethesda assay. Methods Samples from the time of first diagnosis and clinical data were available from 102 patients with acquired hemophilia enrolled in the prospective GTH-AH 01/2010 study. Controls (n = 102) were matched for gender and age. Diagnostic cut-offs were determined by receiver-operator curve analysis. The prognostic value was assessed in 92 of the 102 patients by Cox regression analysis of time to partial remission. Results Anti-FVIII IgG above the 99th percentile (> 15 arbitrary units per mL) revealed high sensitivity and specificity (both 0.99; 95% confidence interval, 0.95-1.0) for diagnosing acquired hemophilia. The likelihood of achieving partial remission was related to anti-FVIII IgG concentration ( 1050, 0.39). The Bethesda titer was only associated with the likelihood of partial remission when analyzed in the central laboratory, but not when data from local GTH study sites were used. Conclusion Although the Nijmegen-modified Bethesda assay is the reference

  15. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  16. Asma y deficiencia de subclases de IgG Asthma and IgG subclases deficiency

    Directory of Open Access Journals (Sweden)

    Lucía Santamaría Ortiz

    1995-04-01

    Full Text Available

    Se estudiaron 45 pacientes asmáticos adultos de difícil manejo, de más de 5 años de evolución, 37 de ellos esteroide dependientes y 8 no dependientes, con asma alérgica o intrínseca y algunos con Infecciones respiratorias recurrentes de predominio viral. Por nefelometría se midieron los niveles séricos de las IgsG, M y A, y por ELISA se determinó la IgE total. Se encontraron 4 pacientes con deficiencia de IgG total, en el grupo de los esteroide dependientes. Mediante ELISA tipo sandwich y con anticuerpos monoclonales específicos para las sub clases de IgG se investigaron los niveles sé ricos de IgG1, 2, 3 y 4. En el 55.6% de los enfermos se encontraron una O más deficiencias de sub clases. No hubo diferencias significativas entre los grupos esteroide y no esteroide dependientes, ni entre los asmáticos alérgicos e intrínsecos, ni entre los con infección recurrente o sin ella. predominó la deficiencia de IgG1; en total el 46.7% de los pacientes tenían deficiencia aislada o combinada de IgG1, el 31.1% de IgG2, el 24.4% de IgG3 y el 17.8% de Igd4. La alta incidencia de deficiencia de sub clases podría deberse a la acción de los esteroides o a una alteración en la regulación de la síntesis de Igs producida por un defecto Inmune primario. Esta deficiencia sería la responsable del comportamiento agresivo de la enfermedad.

    We studied 45 adult asthmatic patients with difficult to care disease and who had more than five years of evolution; they suffered from elther allergic or intrinsic asthma and some had experienced recurrent respiratory tract infections. predominantly of viral etiology. Serum levels of IgA, IgG and IgM were measured by nephelometry and total lgE was determined by an Enzyme-Linked immunosorbent Assay (ELISA. Total lg

  17. Detection of LGI1 and CASPR2 antibodies with a commercial cell-based assay in patients with very high VGKC-complex antibody levels.

    Science.gov (United States)

    Yeo, T; Chen, Z; Chai, J Y H; Tan, K

    2017-07-15

    The presence of VGKC-complex antibodies, without LGI1/CASPR2 antibodies, as a standalone marker for neurological autoimmunity remains controversial. Additionally, the lack of an unequivocal VGKC-complex antibody cut-off level defining neurological autoimmunity makes it important to test for monospecific antibodies. We aim to determine the performance characteristics of a commercial assay (Euroimmun, Lübeck, Germany) for LGI1/CASPR2 antibody detection in patients with very high VGKC-complex antibody levels and report their clinico-serological associations. We identified 8 patients in our cohort with the highest VGKC-complex antibody levels (median 2663.5pM, range 933-6730pM) with VGKC-complex antibody related syndromes (Group A). Two other groups were identified; 1 group with suspected neuronal surface antibody syndromes and negative for VGKC-complex antibodies (Group B, n=8), and another group with cerebellar ataxia and negative for onconeuronal antibodies (Group C, n=8). Seven out of 8 patients (87.5%) in Group A had LGI1 and/or CASPR2 antibodies. One Group B patient had LGI1 antibodies but was negative on re-testing with a live cell assay. No Group C patients had monospecific antibodies. Inter-rater reliability was high; combining Groups A and B patients, the kappa statistic was 0.87 and 1.0 for LGI1 and CASPR2 antibodies respectively. We demonstrated that a high proportion of patients with very high VGKC-complex antibody levels and relevant clinical syndromes have LGI1 and/or CASPR2 antibodies detected by the commercial assay. Our findings lend support to the use of the assay for rapid and reliable detection of LGI1 and CASPR2 antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Absence of antibodies against Bordetella pertussis in pregnant women and newborns in the state of Nuevo Leon.

    Science.gov (United States)

    Villarreal-Pérez, Jesús Zacarías; Ramírez-Aranda, José Manuel; Rodríguez-Rodríguez, Irasema; Perales-Dávila, José; García-Elizondo, Francisco Javier; Gómez-Gómez, Celina; Galindo-Galindo, Edgar; Rodríguez-Moreno, Marco Antonio; Ballesteros-Elizondo, Romelia

    2014-09-01

    To assess placental transfer of antibodies to the child at birth and at 2 months of age. For the quantification of anti-PT IgG antibodies, we used an indirect enzyme-linked immunosorbent assay standardized by The National Institute of Epidemiologic Diagnosis and Reference (InDRE). Samples were considered negative from 0 to 48 IU/mL, indeterminate from 49 to 93 IU/mL and positive at ≥94 IU/mL. We performed a cross-sectional assessment of anti-PT IgG antibody levels in the mother, umbilical cord, and child. There was a higher concentration of IgG anti-TP in the umbilical cord (4.3%) and in the mother (1.4%), but a total absence was observed in the child (0%). The vulnerability of children to Bordetella pertussis shows the need to implement effective immunization strategies, whether actively, in the child, or passively through the mother, adolescents, and adults who are in contact with the child.

  19. Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay

    International Nuclear Information System (INIS)

    Sobotka, A.K.; Valentine, M.D.; Ishizaka, K.; Lichtenstein, L.M.

    1976-01-01

    A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r/sub s/ = 0.96 p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective

  20. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    Science.gov (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

    2016-04-01

    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  1. Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

    DEFF Research Database (Denmark)

    Samuelsen, Simone; Jørgensen, Christian Damsgaard; Mellins, Elizabeth D

    2018-01-01

    In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential...... linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies...... and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p

  2. Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - Prediction of viscosity through protein-protein interaction measurements

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Kalonia, Devendra S; Parshad, Henrik

    2013-01-01

    The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI...... low or high protein concentration determined using DLS. The PPI measurements were correlated with solution viscosity (measured by DLS using polystyrene nanospheres and ultrasonic shear rheology) as a function of pH (4-9) and ionic strength (10, 50 and 150mM). Our measurements showed that the highest...... solution viscosity was observed under conditions with the most negative kD, the highest apparent radius and the lowest net charge. An increase in ionic strength resulted in a change in the nature of the PPI at low pH from repulsive to attractive. In the neutral to alkaline pH region the mAbs behaved...

  3. Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

    Science.gov (United States)

    Miller, C H; Boylan, B; Shapiro, A D; Lentz, S R; Wicklund, B M

    2017-10-01

    Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG 4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG 4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG 4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG 4 in some patients with negative (NBA, supporting the need for

  4. Sensitive and specific detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)—Specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests

    Science.gov (United States)

    Emmerich, Petra; Mika, Angela; von Possel, Ronald; Rackow, Anne; Liu, Yang; Schmitz, Herbert; Sherifi, Kurtesh; Halili, Barie; Jakupi, Xhevat; Berisha, Lindita; Ahmeti, Salih

    2018-01-01

    As the most widespread tick-borne arbovirus causing infections in numerous countries in Asia, Africa and Europe, Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) was included in the WHO priority list of emerging pathogens needing urgent Research & Development attention. To ensure preparedness for potential future outbreak scenarios, reliable diagnostic tools for identification of acute cases as well as for performance of seroprevalence studies are necessary. Here, the CCHFV ortholog of the major bunyavirus antigen, the nucleoprotein (NP), was recombinantly expressed in E.coli, purified and directly labeled with horseradish peroxidase (HRP). Employing this antigen, two serological tests, a μ-capture ELISA for the detection of CCHFV-specific IgM antibodies (BLACKBOX CCHFV IgM) and an IgG immune complex (IC) ELISA for the detection of CCHFV-specific IgG antibodies (BLACKBOX CCHFV IgG), were developed. Test performance was evaluated and compared with both in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available ELISA tests (VectoCrimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples collected in Kosovo during the years 2013–2016 from 15 patients with an acute, RT-PCR-confirmed CCHFV infection, and 12 follow-up sera of the same patients collected approximately one year after having overcome the infection. Reliably detecting IgM antibodies in all acute phase sera collected later than day 4 after onset of symptoms, both IgM ELISAs displayed excellent diagnostic and analytical sensitivity (100%, 95% confidence interval (CI): 85.2%–100.0%). While both IgG ELISAs readily detected the high IgG titers present in convalescent patients approximately one year after having overcome the infection (sensitivity 100%, 95% CI: 73.5%–100.0%), the newly developed BLACKBOX CCHFV IgG ELISA was superior to the commercial IgG ELISA in detecting the rising IgG titers during the acute phase

  5. IgG/IgM antiphospholipid antibodies present in the classification criteria for the antiphospholipid syndrome: a critical review of their association with thrombosis.

    Science.gov (United States)

    Kelchtermans, H; Pelkmans, L; de Laat, B; Devreese, K M

    2016-08-01

    Essentials The clinical value of IgM antibodies in thrombotic antiphospholipid syndrome (APS) is debated. By review of literature, we reconsidered the clinical value of IgM antibodies in thrombotic APS. More significant correlations with thrombosis were found for the IgG compared to IgM isotype. Unavailability of paired IgG/IgM results hampers evaluating the added value of IgM positivity. Click to hear Dr de Groot's perspective on antiphospholipid syndrome Background Despite the update of the classification criteria for the antiphospholipid syndrome (APS), difficulties persist in the identification of patients at risk for thrombosis. Current guidelines include assays detecting IgG/IgM anti-β2 -glycoprotein I and anti-cardiolipin antibodies, although the relevance of IgM antibodies has been debated. Objectives Through a review of the literature from 2001 to 2014, we aimed to formally establish the thrombotic risk stratification potential of IgM as compared with IgG anti-phospholipid antibodies (aPLs). Patients/methods One thousand two hundred and twenty-eight articles were selected by a computer-assisted search of the literature. Of the 177 studies that met our inclusion criteria, the clinical value of IgG/IgM aPLs was established through analysis of odds ratios for thrombosis or percentage of positives in the thrombotic population. Results/conclusions We clearly found more significant correlations with thrombosis for the IgG than for the IgM isotype. Nonetheless, in a minority of studies, significant associations with thrombosis were found for IgM but not IgG antibodies. The unavailability of paired results of IgG and IgM for each separate patient hampers evaluation of the added value of isolated IgM positivity. To fully take advantage of results obtained by future studies, we strongly encourage scientists to provide all studied information per patient. We planned a large multicenter study to investigate clinical associations of isolated/combined positivity for

  6. Effect of serum heat-inactivation and dilution on detection of anti-WNV antibodies in mice by West Nile virus E-protein microsphere immunoassay.

    Directory of Open Access Journals (Sweden)

    Madhuri Namekar

    Full Text Available Immunopathogenesis studies employing West Nile virus (WNV mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.

  7. Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic.

    Science.gov (United States)

    Bogdanos, Dimitrios-Petrou; Baum, Harold; Okamoto, Manabu; Montalto, Paolo; Sharma, Umesh C; Rigopoulou, Eirini I; Vlachogiannakos, John; Ma, Yun; Burroughs, Andrew K; Vergani, Diego

    2005-08-01

    The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti-PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]AE shared by PDC-E2(212-226) and hsp's is a cross-reactive target. Having found that this same motif is present only in beta-galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from 100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia coli (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL(266-280) LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57%) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12%; P ECOLI PDC-E2 reactivity was virtually absent. BGAL(266-280)/PDC-E2(212-226) reactivity of the IgG3 isotype was found in 52 (52%) AMA-positive PBC patients but in only 1 of the controls (P ECOLI PDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC.

  8. Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

    Science.gov (United States)

    Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

    1988-01-01

    Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

  9. Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

    Science.gov (United States)

    Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

    2009-04-01

    The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

  10. Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study.

    Science.gov (United States)

    Islam, Sidra; Moinuddin; Mir, Abdul Rouf; Raghav, Alok; Habib, Safia; Alam, Khursheed; Ali, Asif

    2017-09-12

    Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

  11. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

    OpenAIRE

    Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

    2017-01-01

    Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

  12. Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom Los anticuerpos IgG de conejos anti-fosfolipasa A2 de Crotalus durissus terrificus neutralizan la actividad letal del veneno

    Directory of Open Access Journals (Sweden)

    Juan P. Rodríguez

    2006-12-01

    Full Text Available Crotalus durissus terrificus (C.d.t. (South American rattlesnake venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2 and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75. Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg with Freund adjuvant. Groups of six mice (20 + 2 g were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.El veneno de Crotalus durissus terrificus (C.d.t. (Cascabel de Sud América posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2 y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75. Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del

  13. Immunoglobulin G4: an odd antibody

    NARCIS (Netherlands)

    Aalberse, R. C.; Stapel, S. O.; Schuurman, J.; Rispens, T.

    2009-01-01

    Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

  14. The apparent monovalency of human IgG4 is due to bispecificity

    NARCIS (Netherlands)

    Aalberse, R. C.; Schuurman, J.; van Ree, R.

    1999-01-01

    A hypothesis is put forward to explain the apparent monovalency of human IgG4. It is based upon the known instability of the IgG4 hinge. IgG4 is secreted as a regular bivalent antibody, but after secretion interacts with another IgG4 molecule. This interaction results in the exchange of half

  15. Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

    Science.gov (United States)

    Igawa, Tomoyuki

    2017-01-01

    Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

  16. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  17. Varicella zoster virus myelitis in two elderly patients: diagnostic value of nested polymerase chain reaction assay and antibody index for cerebrospinal fluid specimens.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Miki, Kenji; Yamaguchi, Mai; Kanno, Akira; Nunomura, Satoshi; Ra, Chisei; Tamiya, Takashi; Kamei, Satoshi; Takasu, Toshiaki

    2013-01-01

    Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV) infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th)-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR) assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF) specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

  18. Isotypes of Epstein-Barr Virus Antibodies in Rheumatoid Arthritis: Association with Rheumatoid Factors and Citrulline-Dependent Antibodies

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    Marie Wulff Westergaard

    2015-01-01

    Full Text Available In order to study the humoral immune response against Epstein-Barr virus (EBV in patients with rheumatoid arthritis (RA and to compare it with the two major autoantibody types in RA, plasma samples from 77 RA patients, 28 patients with systemic lupus erythematosus (SLE, and 28 healthy controls (HCs were investigated by enzyme-linked immunosorbent assays (ELISA. Increased percentages of positives and concentrations of IgG/IgA/IgM antibodies against the latent EBV nuclear antigen-1 (EBNA-1 were observed in RA patients compared to SLE patients and HCs. Increased concentrations and percentages of positives of IgG/IgA/IgM against the early lytic EBV antigen diffuse (EAD were also found in RA patients compared to HCs but were highest in SLE patients. Furthermore, associations between the elevated EBNA-1 IgA and EBNA-1 IgM levels and the presence of IgM and IgA rheumatoid factors (RFs and anti-citrullinated protein antibodies (ACPAs, IgG and between elevated IgA concentrations against EAD and the presence of RFs and ACPAs in RA patients were found. Thus, RA patients had elevated antibodies of all isotypes characteristic of latent EBV infection (whereas SLE patients had elevated antibodies characteristic of lytic EBV infection. Notably, for IgM and IgA (but not IgG, these were associated with the presence of characteristic RA autoantibodies.

  19. Falsely low immunoglobulin (Ig)G4 in routine analysis: how not to miss IgG4 disease.

    Science.gov (United States)

    Egner, W; Swallow, K; Lock, R J; Patel, D

    2016-10-01

    Immunoglobulin (Ig)G4 disease can have apparently 'normal' levels of IgG4 due to antigen excess conditions. IgG4 measurement therefore appears falsely low. UK National External Quality Assurance Scheme (UK NEQAS) data and other reports have suggested that this problem occurred despite pre-existing antigen excess detection steps. To determine the clinical relevance of the problem, we examined the prevalence and characteristics of prozoning in our laboratory and patient cohorts. We establish that the prevalence of raised IgG4 in routine IgG4 analysis is low (IgG4 samples in our patients. This may explain the previous reports of low sensitivity of raised IgG4 for IgG4RD, and predictive values should be re-evaluated in this disease using modified prozone-resistant protocols. All laboratories providing IgG4 measurements should verify that their assays are fit for the clinical quality requirement of detection raised IgG4 levels and must verify the upper limit of their reference ranges and freedom from prozoning. © 2016 British Society for Immunology.

  20. Highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D. (Hebrew Univ., Jerusalem (Israel). Hadassah Medical School)

    1982-08-27

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10/sup 6/ RBC.

  1. Anti-phospholipid antibodies in patients with Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Jakobsen, P H; Morris-Jones, S D; Hviid, L

    1993-01-01

    Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types...... of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were...... significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease....

  2. Prevalence of serum antibodies to hantavirus in a rural population from the southern state of Santa Catarina, Brazil

    Directory of Open Access Journals (Sweden)

    Gregório Wrublevski Pereira

    2012-02-01

    Full Text Available INTRODUCTION: Rodent-borne hantaviruses cause severe human diseases. We completed a serological survey of hantavirus infection in rural inhabitants of Turvo County, in the southern State of Santa Catarina, Brazil, in which seropositivity for hantavirus was correlated to previous disease in the participants. METHODS: The levels of IgG antibodies to hantavirus Araraquara in the sera of 257 individuals were determined using an immunoenzymatic assay. RESULTS: IgG antibodies to hantavirus were found in 2.3% of the participants. All seropositive participants reported previous disease with symptoms suggestive of hantavirus cardiopulmonary syndrome. CONCLUSIONS: Human infections causing unreported cardiopulmonary syndrome probably occur in the southern State of Santa Catarina.

  3. Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

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    Haddad Muhammad

    2016-01-01

    Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

  4. Prevalence of IgG varicella zoster virus antibodies in the Kuikuro and Kaiabi indigenous communities in Xingu National Park, Brazil, before varicella vaccination Prevalência de anticorpos IgG contra o vírus varicela zoster nas aldeias indígenas Kuikuro e Kaiabi do Parque Nacional do Xingu, Brasil, antes da vacinação contra varicela

    Directory of Open Access Journals (Sweden)

    Manuel Mindlin Lafer

    2005-06-01

    Full Text Available The purpose of the study was to estimate the prevalence of IgG antibodies against varicella zoster virus (VZV in the two most populated indigenous ethnic groups from Xingu Indigenous National Park, in Brazil, prior to the introduction of vaccination against the disease, and to determine the positive and the negative predictive values of a history of varicella infection. In 2001, 589 inhabitants of two Kuikuro villages and three Kaiabi villages were evaluated and provided information concerning previous varicella infection. An indirect immunosorbent assay (ELISA to detect IgG anti-VZV antibodies was performed in 224 blood samples - volunteer selection had no interference of anamnesis. IgG prevalence was 80.8% (95% Confidence Interval: 76% - 86%. The seroepidemiology of varicella in Xingu National Park prior to varicella vaccine introduction was comparable to the Brazilian national seroprevalence described in the literature, and so were the positive (98% and the negative predictive value (41% of the referred history.O objetivo do estudo foi aferir a prevalência de anticorpos IgG contra o Vírus Varicela-Zoster (VVZ nos dois grupos étnicos indígenas mais povoados do Parque Nacional Indígena do Xingu, Brasil, antes da introdução da vacinação contra a doença, e determinar os valores preditivos positivo e negativo da história de infecção de varicela. Em 2001, 589 habitantes de duas aldeias Kuikuro e três aldeias Kaiabi foram avaliados e forneceram dados referentes à infecção prévia por varicela. Um ensaio imunoenzimático indireto (ELISA foi realizado em 224 amostras de sangue para detectar anticorpos IgG anti-VVZ - a seleção de voluntários não teve interferência da anamnese. A prevalência de IgG foi de 80,8% (Intervalo de Confiança de 95%: 76% - 86%. A soroepidemiologia de varicela no Parque Nacional do Xingu antes da introdução da vacina foi comparável à soroprevalência nacional descrita na literatura, assim como os

  5. Estimation of polyclonal IgG4 hybrids in normal human serum.

    Science.gov (United States)

    Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

    2014-07-01

    The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. © 2014 John Wiley & Sons Ltd.

  6. Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

    Directory of Open Access Journals (Sweden)

    Rafaella Fortini Queiroz Grenfell

    2012-08-01

    Full Text Available INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62. In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively, whereas ELISA using egg antigens (ELISA-SEA detected samples after 140 days (p=0.03. CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

  7. Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis

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    Kazutaka Kitaura

    2017-05-01

    Full Text Available A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM. Class-switch recombination (CSR produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V, junctional (J sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system.

  8. Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

    Science.gov (United States)

    Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

    1999-01-01

    The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

  9. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1982-01-01

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG 1 subclass. Affinities of antibodies for TSH were in the range 2 x 10 8 -5 x 10 10 M -1 . Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  10. Behavioral and neural effects of intra-striatal infusion of anti-streptococcal antibodies in rats

    Science.gov (United States)

    Lotan, Dafna; Benhar, Itai; Alvarez, Kathy; Mascaro-Blanco, Adita; Brimberg, Lior; Frenkel, Dan; Cunningham, Madeleine W.; Joel, Daphna

    2014-01-01

    Group A β-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and 5HT-2A and 5HT-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders. PMID:24561489

  11. Serum Dried Samples to Detect Dengue Antibodies: A Field Study

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    Angelica Maldonado-Rodríguez

    2017-01-01

    Full Text Available Background. Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs. Methods. Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. Results. DSS elution conditions were standardized as follows: 1 h at 4°C in 200 µl of DNase-, RNase-, and protease-free PBS (1x. The optimal volume of DSS eluate to be used in the IgG assay was 40 µl. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. Conclusion. DSS samples are useful for detecting anti-dengue IgG antibodies in the field.

  12. Food related antibodies in headache patients.

    OpenAIRE

    Merrett, J; Peatfield, R C; Rose, F C; Merrett, T G

    1983-01-01

    Highly sensitive and specific methods for assaying IgE and IgG4 for antibodies in serum have been developed in order to test a recent suggestion that food allergy is a major cause of migraine. Sera were collected from 208 adults--74 with dietary migraine, 45 with non-dietary migraine, 29 with cluster headache and 60 controls. No significant differences were identified between any of the groups with the one exception that cluster headache patients had significantly raised levels of total serum...

  13. Assessment of IgG Antibodies Against HSV1, HSV2, CMV and EBV in Patients with Pemphigus Vulgaris versus Healthy People

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    Parichehr Ghalayani

    2016-08-01

    Full Text Available Objectives: Regarding the implication of viruses particularly herpes in pemphigus vulgaris, we sought to assess and compare the level of immunoglobulin G (IgG antibodies against herpes simplex virus types 1 and 2 (HSV1 and HSV2, cytomegalovirus (CMV and Epstein-Barr virus (EBV in patients with pemphigus vulgaris and healthy people.    Materials and Methods: In this cross-sectional study, 25 patients with pemphigus vulgaris and 27 healthy individuals comprised the experimental and control groups, respectively. Serum samples were taken from both groups; the levels of IgG antibodies against HSV1, HSV2, CMV and EBV were measured using ELISA.  Results: Immunoglobulin G titer was higher for all four viruses in the patient group in comparison to the control group. This difference was significant for anti-EBV (P= 0.005, anti-CMV (P=0.0001 and anti-HSV2 (P=0.001 but not significant for anti-HSV1 (P= 0.36.Conclusion: Viruses including EBV, CMV, and HSV2 probably play a role in the pathogenesis of pemphigus in addition to the effects of genetics, toxins and other predisposing factors. In this study, no statistically significant relationship was observed between HSV1 and pemphigus vulgaris, which was probably due to the high titer of anti-HSV1 IgG in healthy individuals in the community. More studies must be done in this regard.

  14. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

    Directory of Open Access Journals (Sweden)

    Chao Shan

    2017-03-01

    Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  15. The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

    Directory of Open Access Journals (Sweden)

    Christine Rasetti-Escargueil

    2017-10-01

    Full Text Available The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies. For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs. The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

  16. Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens.

    Directory of Open Access Journals (Sweden)

    Nancy O Duah

    2009-10-01

    Full Text Available It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types and MSP3 (two allelic types. Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h(2 estimates for 48% (20/42 of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested among the adults, 48% (20/42 among the children in the dry season and 31% (13/42 among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20 of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.

  17. Antiphosphatidylserine/prothrombin antibodies as biomarkers to identify severe primary antiphospholipid syndrome.

    Science.gov (United States)

    Hoxha, Ariela; Mattia, Elena; Tonello, Marta; Grava, Chiara; Pengo, Vittorio; Ruffatti, Amelia

    2017-05-01

    Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies have begun to be considered potentional biomarkers for antiphospholipid syndrome (APS). This cohort study investigate the role of aPS/PT antibodies as a risk factor for severe APS by evaluating the association between those antibodies and clinical/laboratory profiles of APS. Plasma/serum samples from 197 APS patients, 100 healthy subjects and 106 patients with autoimmune diseases were collected. IgG/IgM aPS/PT antibodies were assayed using commercial ELISA kit. Prevalences of IgG and IgM aPS/PT (pantiphospholipid antibody patients than in double and single positivity ones (p<0.0001 for all). APS/PT antibodies were associated to severe thrombosis, severe pregnancy complications inducing prematurity, and vascular microangiopathy, all generally associated to high risk APS forms requiring strong therapy.

  18. Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.

    Directory of Open Access Journals (Sweden)

    Christian Roussilhon

    2007-11-01

    Full Text Available BACKGROUND: Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite to molecules that are considered as valuable vaccine candidates. METHODS AND FINDINGS: We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults. The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3, was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week. Finally, the target epitopes of these antibodies were found to be fully conserved. CONCLUSIONS: Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be

  19. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    OpenAIRE

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-01-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems...

  20. Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

    Science.gov (United States)

    Díaz-Solano, Dylana; Fuenmayor, Jaheli; Montaño, Ramon F

    2018-02-01

    Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. Recombinant polymeric anti-G IgG1 (IgG1μtp) and IgG3 (IgG3μtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. The recombinant IgG1μtp and IgG3μtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. The enhanced opsonic properties of the polymeric anti-G IgG1μtp and IgG3μtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

  1. Performance of an ELISA and Indirect Immunofluorescence Assay in Serological Diagnosis of Zoonotic Cutaneous Leishmaniasis in Iran

    Directory of Open Access Journals (Sweden)

    Bahador Sarkari

    2014-01-01

    Full Text Available Serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (VL and considered as a routine method for diagnosis of VL while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (CL. This study aimed to assess the performance of indirect immunofluorescent-antibody test (IFA and enzyme-linked immunosorbent assay (ELISA for serodiagnosis of cutaneous leishmaniasis in Iran. Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera from healthy controls along with 50 sera from non-CL patients were collected. Antigen was prepared from promastigotes and amastigotes of Leishmania major. IFA was used to detect anti-Leishmania IgG while ELISA was used to detect anti-Leishmania IgM, total IgG, or IgG subclasses (IgG1 and 4. ELISA, for detection of total IgG and IgM, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. Sensitivity and specificity of ELISA for detecting IgG1 and IgG4 were 64%, 75% and 85%, 49%, respectively. Sensitivity and specificity of IFA were 91.6% and 81%. Conclusion. Findings of this study demonstrated that serological test, especially IFA, can be used for proper diagnosis of CL.

  2. Interference by antiruthenium antibodies in the Roche thyroid-stimulating hormone assay

    NARCIS (Netherlands)

    Buijs, M. M.; Gorgels, J. P. M. C.; Endert, E.

    2011-01-01

    There are many causes of interference in immunoassays causing erratic patient results. A method-specific interference due to antiruthenium antibodies in Roche free thyroxine (fT4) and free triiodothyronine (fT3) assays has been described previously. As a result, a new generation fT4 assay has been

  3. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    Cowan, S.I.; Stagg, B.H.; Niemann, E.

    1977-01-01

    Variations in the sub-unit antigenic structure of ferritins derived from various human tissues are reflected in the differing specificities of antisera raised against these ferritin preparations. In this study it was shown that antibody specificity played an important role in determining the sensitivity and overall binding of labelled antibody in a two-site immunoradiometric assay for ferritin. Homologous assay systems, in which solid phase and radiolabelled antibodies were of similar specificities, were generally less sensitive and showed lower binding than heterologous assay systems, in which solid phase and labelled antibodies were of different specificities. The source of the ferritin which was used as assay standard also played an important part in determining the sensitivity and overall binding in homologous antibody systems, spleen ferritin standards yielding assays superior to those obtained with placenta or liver ferritin standards. However, these differences between standards were not seen in a heterologous system employing solid phase antibodies directed against liver ferritin and labelled antibodies directed against placenta ferritin. The nature of the ferritin used to prepare immunoadsorbant for the purification of antibodies prior to radioiodination also affected the assay characteristics; antibodies prepared on spleen ferritin immunoadsorbant being more reactive than antibodies prepared on placenta ferritin immunoadsorbant, which in turn were more reactive then antibodies prepared on liver ferritin immunoadsorbant. (orig.) [de

  4. Fc-Glycosylation in Human IgG1 and IgG3 Is Similar for Both Total and Anti-Red-Blood Cell Anti-K Antibodies

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    Myrthe E. Sonneveld

    2018-01-01

    Full Text Available After albumin, immunoglobulin G (IgG are the most abundant proteins in human serum, with IgG1 and IgG3 being the most abundant subclasses directed against protein antigens. The quality of the IgG-Fc-glycosylation has important functional consequences, which have been found to be skewed toward low fucosylation in some antigen-specific immune responses. This increases the affinity to IgG1-Fc-receptor (FcγRIIIa/b and thereby directly affects downstream effector functions and disease severity. To date, antigen-specific IgG-glycosylation have not been analyzed for IgG3. Here, we analyzed 30 pregnant women with anti-K alloantibodies from a prospective screening cohort and compared the type of Fc-tail glycosylation of total serum- and antigen-specific IgG1 and IgG3 using mass spectrometry. Total serum IgG1 and IgG3 Fc-glycoprofiles were highly similar. Fc glycosylation of antigen-specific IgG varied greatly between individuals, but correlated significantly with each other for IgG1 and IgG3, except for bisection. However, although the magnitude of changes in fucosylation and galactosylation were similar for both subclasses, this was not the case for sialylation levels, which were significantly higher for both total and anti-K IgG3. We found that the combination of relative IgG1 and IgG3 Fc-glycosylation levels did not improve the prediction of anti-K mediated disease over IgG1 alone. In conclusion, Fc-glycosylation profiles of serum- and antigen-specific IgG1 and IgG3 are highly similar.

  5. Production of double antibody for radioimmunoassay (sheep anti-rabbit IgG antiserum); Producao de duplo anticorpo para radioimunoensaio (antissoro de carneiro anti-IgG de coelho)

    Energy Technology Data Exchange (ETDEWEB)

    Silva, S.R. da

    1994-12-31

    A second antibody (sheep anti-rabbit IgG antiserum) to be used in RIAs in which the first antibody is raised in rabbits was produced. For this production, initially the IgG was isolated from rabbit serum and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. Four sheep were immunized with 500 u g of purified rabbit IgG, emulsified in Freund Complete Adjuvant and administered by multisite subcutaneous injections. These injections were repeated at 20-days intervals and blood samples (40 ml) were taken from the jugular vein 10 days after the boosts for the evaluation of the antisera title. After each four boosts a great bleeding was done by the same route. Approximately 500 ml of serum were obtained in each bleeding per animal. The antisera were evaluated by the human thyrotropin RIA developed at IPEN laboratories employing reagents provided by NIDDKD, USA. These evaluations referred to the determination of the antisera title and of the ideal concentration of carrier IgG, to the study of the kinetic of precipitation and to the confirmation of the inexistent cross-reactivity with human IgG, in comparison with a reference antiserum of know precipitation characteristics supplied by the Radioassay System Laboratories. Approximately 3,6 l of antiserum (sheep anti-rabbit IgG serum) were produced from the four sheep, which presented title and precipitation characteristics very similar to those exhibited by the imported commercial product, even presenting higher titles. The results obtained in this work indicated that it was created enough experience for the production of this biological reagent for RIA, that could be done integrally in the country in greater scale, and at a very reduced cost. (author). 81 refs, 36 figs, 33 tabs.

  6. Duration of detection of anti-BmR1 IgG4 antibodies after mass-drug administration (MDA) in Sarawak, Malaysia.

    Science.gov (United States)

    Noordin, R; Muhi, J; Md Idris, Z; Arifin, N; Kiyu, A

    2012-03-01

    The detection rates of brugian filariasis in three regions of Sarawak namely Central, North and South after three courses of mass drug administration (MDA) from year 2004 to 2006 was investigated. A recombinant BmR1 antigen-based IgG4 detection test, named Brugia Rapid and night blood smear for microfilaria (mf) detection were used. All three regions recorded a sharp fall in mf positive rates after a year post-MDA. Meanwhile Brugia Rapid positive rates declined more gradually to 3.8% and 5.6% of the pre-MDA levels in the Central and North regions, respectively. This study showed that in filariasis endemic areas in Sarawak, anti-filarial IgG4 antibodies to BmR1, as detected by the Brugia Rapid test, were positive for one to two years after mf disappearance.

  7. Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum.

    Science.gov (United States)

    Lee, Hyunju; Lim, Soo Young; Kim, Kyung Hyo

    2017-10-01

    The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05-0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. © 2017 The Korean Academy of Medical Sciences.

  8. A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    International Nuclear Information System (INIS)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D.

    1982-01-01

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10 6 RBC. (Auth.)

  9. IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

    Science.gov (United States)

    Wiertsema, Selma P; Corscadden, Karli J; Mowe, Eva N; Zhang, Guicheng; Vijayasekaran, Shyan; Coates, Harvey L; Mitchell, Timothy J; Thomas, Wayne R; Richmond, Peter C; Kirkham, Lea-Ann S

    2012-01-01

    Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay. Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

  10. IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

    Directory of Open Access Journals (Sweden)

    Selma P Wiertsema

    Full Text Available BACKGROUND: Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. METHODS: We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply and 3 NTHi (P4, P6 and PD proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion and 63 healthy age-matched controls, using a newly developed multiplex bead assay. RESULTS: Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. CONCLUSIONS: Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

  11. Solid phase radioimmunoassays using labelled antibodies: a conceptual framework for designing assays

    International Nuclear Information System (INIS)

    Kalmakoff, J.; Parkinson, A.J.; Crawford, A.M.; Williams, B.R.G.

    1977-01-01

    A simple theoretical model for the antigen-antibody reaction is presented and used to evaluate the optimum conditions for designing solid phase radioimmunoassays (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA described include the direct, the indirect, the direct sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity(>1), and that the formation of the antigen-antibody complex is dependent on the mass action effect

  12. Effects of gamma radiation immunogenicity of ribonucleoprotein (RNPs) of rabies virus and purification of anti-RNPs antibodies for diagnosis

    International Nuclear Information System (INIS)

    Costa, Ana Elena Boamorte da

    2010-01-01

    The World Health Organization recommends the direct immunofluorescence test for laboratory diagnosis and serological evaluation of rabies. To achieve this test, fluorescent anti-ribo nucleoproteins (RNPs) conjugates, produced from purified IgGs of RNP-immunized animals are employed. The aims of the present study were: investigate the effects of gamma radiation on the immunogenicity of RNPs, as well as to compare two chromatographic methodologies for the purification of anti-RNPs immunoglobulins. Sera from animals immunized with either native or irradiated RNPs were compared by direct immunofluorescence and immuno enzymatic assays. Our results indicate that the animals immunized with irradiated antigen requested a lower number of doses to reach high antibody titers. The immunofluorescence assays indicated that the conjugates produced with the anti-irradiated RNPs IgGs showed similar specificity to its anti-native counterpart, but with a higher definition of the virus inclusions. The purification methods were compared by Bradford and electrophoresis assays. According to the results, we concluded that the affinity-based process resulted in higher yields, lower execution time, and higher purity of the antibodies. (author)

  13. Characteristics of primary Sjögren's syndrome patients with IgG4 positive plasma cells infiltration in the labial salivary glands.

    Science.gov (United States)

    Liu, Chang; Zhang, Huayong; Yao, Genhong; Hu, Yunxia; Qi, Jingjing; Wang, Yan; Chen, Weiwei; Tang, Xiaojun; Li, Wenchao; Lu, Liwei; Gu, Luo; Sun, Lingyun

    2017-01-01

    The purpose of this study was to investigate the characteristics of primary Sjögren's syndrome (pSS) patients with IgG4 positive (IgG4 + ) plasma cell infiltration in labial salivary glands (LSGs). Paraffin sections of LSGs from 336 pSS patients were stained with IgG4 and IgG monoclonal antibodies. According to the infiltration of IgG4 + plasma cells, patients were divided and clinical and serological characteristics were analyzed and compared. Based on the infiltration of IgG4 + plasma cells in the LSGs, patients were divided into three subgroups, low IgG4, moderate IgG4, and high IgG4 groups. A negative association between the number of infiltrated IgG4 + plasma cells and the disease characteristics was observed. We found that the higher the IgG4 + expression in plasma cells, the lower the positive rates of serum anti-SSA antibodies, anti-SSB antibodies, antinuclear antibodies (ANA), and rheumatoid factor (RF). Besides, patients from the high IgG4 group had the highest frequency of interstitial lung disease (ILD, 30.6%) and tubulointerstitial nephritis (TIN, 13.9%), but the lowest frequency of leucopenia (13.9%), thrombocytopenia (11.1%), and abnormal thyroidal function (0%). PSS patients with different IgG4 + plasma cells infiltration in the LSGs had distinctive clinical and laboratory characteristics. It may help us to further understand the role of IgG4 + plasma cells in pSS.

  14. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    Science.gov (United States)

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  15. Study of a Cohort of 1,886 Persons To Determine Changes in Antibody Reactivity to Borrelia burgdorferi 3 Months after a Tick Bite

    DEFF Research Database (Denmark)

    Dessau, Ram B; Fryland, Linda; Wilhelmsson, Peter

    2015-01-01

    or relative change is defined by dividing the measurement units from the second sample by those from the first sample. The threshold for the minimum level of significant change was defined at the 2.5% level to represent the random error level. The thresholds were a 2.7-fold rise for the flagellar IgG assay......Lyme borreliosis is a tick-borne disease caused by the bacterium Borrelia burgdorferi. The most frequent clinical manifestation is a rash called erythema migrans. Changes in antibody reactivity to B. burgdorferi 3 months after a tick bite are measured using enzyme-linked immunosorbent assays...... (ELISAs). One assay is based on native purified flagellum antigen (IgG), and the other assay is based on a recombinant antigen called C6 (IgG or IgM). Paired samples were taken at the time of a tick bite and 3 months later from 1,886 persons in Sweden and the Åland Islands, Finland. The seroconversion...

  16. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

    Science.gov (United States)

    Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

    2011-10-28

    Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

    Science.gov (United States)

    Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

    2013-08-01

    The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

  18. Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

    Science.gov (United States)

    Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

    2014-07-17

    Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

    Directory of Open Access Journals (Sweden)

    Camila Zanluca

    Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

  20. ANTIBODIES TO LEUKEMIA DIFFERENTIATION FACTOR (HLDF IN PATIENTS WITH GASTROINTESTINAL CANCER

    Directory of Open Access Journals (Sweden)

    A. I. Autenshlus

    2010-01-01

    Full Text Available Antibodies to leukemia differentiation factor (HLDF in patients with gastrointestinal cancer Abstract. Patients with gastric cancer exhibit higher levels of IgG4-antibodies to human leukemia differentiation factor (HLDF, as compared with healthy individuals, whereas, in patients with colorectal cancer, one may detect high levels of IgA anti-HDLF antibodies, along with lower levels of IgG1 class antibodies against HLDF than in control group. Among patients with gastrointestinal cancer, a positive correlation is revealed between contents of highly differentiated cells in the tumor, and IgM antibodies to HDLF. Meanwhile, a reverse relationship is noted between low differentiation of tumor cells and levels of IgG2 antibodies to HDLF in gastric cancer patients, or IgG3 antibodies to HDLF in patients with colorectal cancer.

  1. Hybrid kappa\\lambda antibody is a new serological marker to diagnose autoimmune pancreatitis and differentiate it from pancreatic cancer.

    Science.gov (United States)

    Hao, Mingju; Li, Wenli; Yi, Lang; Yu, Songlin; Fan, Gaowei; Lu, Tian; Yang, Xin; Wang, Guojing; Zhang, Dong; Ding, Jiansheng; Zhang, Kuo; Zhang, Rui; Lin, Guigao; Han, Yanxi; Wang, Lunan; Li, Jinming

    2016-06-08

    The only generally accepted serological marker currently used for the diagnosis of autoimmune pancreatitis (AIP) is IgG4. Our aim was mainly to determine whether hybrid κ\\λ antibody can help to diagnose AIP and to differentiate it from pancreatic cancer. We established an enzyme-linked immunosorbent assay (ELISA) system to measure the levels of hybrid κ\\λ antibodies in human sera. Sera were obtained from 338 patients, including 61 with AIP, 74 with pancreatic cancer, 50 with acute pancreatitis, 40 with ordinary chronic pancreatitis, 15 with miscellaneous pancreatic diseases, and 98 with normal pancreas. Our study showed levels of hybrid κ\\λ antibodies in the AIP group were significantly higher than in the non-AIP group (P < 0.001). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of AIP were 80.3%, 91%, 66.2% and 95.5% respectively. Furthermore, the combined measurement of serum hybrid κ\\λ antibody and IgG4 tended to increase the sensitivity although the difference was not statistically significant (90.2% vs. 78.7%, P = 0.08), compared to measurement of IgG4 alone. Our findings suggest that hybrid κ\\λ antibody could be a new serological marker to diagnose AIP and differentiate it from pancreatic cancer.

  2. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    International Nuclear Information System (INIS)

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F.

    1990-01-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions

  3. [A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus].

    Science.gov (United States)

    Liu, Lin; Zhang, Quan-Fu; Li, Chuan; Li, Jian-Dong; Jiang, Xiao-Lin; Zhang, Fu-Shun; Wu, Wei; Liang, Mi-Fang; Li, De-Xin

    2013-06-01

    To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA. A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method. The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949). The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.

  4. Immunoradiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs

    International Nuclear Information System (INIS)

    Ahlstedt, S.; Rylander, H.

    1975-01-01

    An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum, 125 I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The antibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. The variability of the assay as judged from the difference between duplicate samples was found to be 18 percent+-4 (p<0.01) of the mean value and the variability between the same serum ran on different test occasions 13 percent+-7 (p<0.01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments. Preincubation of the antisera with dental plaque antigen significantly inhibited the antigen-antibody reaction in the IRMA, while bovine serum albumin did not. (author)

  5. Immunoradiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs

    Energy Technology Data Exchange (ETDEWEB)

    Ahlstedt, S; Rylander, H [Goeteborg Univ. (Sweden)

    1975-01-01

    An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum, /sup 125/I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The antibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. The variability of the assay as judged from the difference between duplicate samples was found to be 18 percent+-4 (p<0.01) of the mean value and the variability between the same serum ran on different test occasions 13 percent+-7 (p<0.01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments. Preincubation of the antisera with dental plaque antigen significantly inhibited the antigen-antibody reaction in the IRMA, while bovine serum albumin did not.

  6. Kinetics of IgG antibody to cytomegalovirus (CMV) after birth and seroprevalence of anti-CMV IgG in Chinese children.

    Science.gov (United States)

    Chen, Jie; Hu, Lingqing; Wu, Meiling; Zhong, Tianying; Zhou, Yi-Hua; Hu, Yali

    2012-12-10

    Prevalence of cytomegalovirus (CMV) infection is 90-100% in developing countries; however, the kinetics of anti-CMV IgG in infants remains elusive. Sera from 112 mother-newborn pairs and longitudinal samples from 41 infants up to 2-year old were tested for anti-CMV IgG and IgM. Additionally, samples from 837 healthy children were included. Of 112 mothers, 108 (96.4%) were anti-CMV IgG positive; their 108 newborns were also seropositive. In a 2-year follow-up among 40 infants of positive mothers, anti-CMV IgG level in 8 individuals decreased with time and became undetectable by age of 3.5-8 months, and that in 32 others decreased at 1- and 3.5-month old, and then increased. Based on the positive IgM, rising IgG levels, and low anti-CMV IgG avidity index, 76.7% of the primary infections were demonstrated to occur during 1-3.5 months of age. The overall seroprevalence of anti-CMV in 837 children was 82.4%, which was generally constant from 2 to 8 years old (χ2 = 3.150, p = 0.790). The maternally acquired anti-CMV IgG in infants disappears before 8-month old. Primary CMV infection in Chinese children mostly occurs during 1-3.5 months of age. Whether the relatively lower seroprevalence of anti-CMV in Chinese children found in this survey may reflect the positive rate in child-bearing age women in the future remains to be further studied.

  7. Comparison of Salivary Anti Helicobacter pylori IgG with Serum IgG and Bacteriological Tests in Detecting Helicobacter pylori Infections

    Directory of Open Access Journals (Sweden)

    H Ghasemian safaei

    2005-01-01

    Full Text Available Background: This study was conducted to compare the efficacy of enzyme-linked immunosorbent assay (ELISA for detecting anti-Helicobacter pylori (H. pylori specific IgG antibodies in specimens of oral fluid and serum with bacteriological tests. Methods: Antral biopsy specimens, as well as serum and oral fluid samples were collected from 97 patients who underwent upper gastrointestinal endoscopy. The presence or absence of current H. pylori infection was determined by culture, histology and urease detection. Anti-H. pylori specific IgG was detected in serum and oral fluid, using an established lab-made, and a commercial ELISA kit. The obtained data were compared with results of bacteriological tests. Results: In all, 62 (64% of 97 patients were positive for H. pylori by one or more of the gold standard tests (culture, histology and urease detection. Lab-made enzyme-linked immunoassay of oral fluid had a sensitivity and specificity of 92% and 83% respectively. A sensitivity and specificity of 87% and 83%, respectively, was obtained with the commercial kit. Lab-made enzyme-linked immunoassay of serum samples had a sensitivity and specificity of 90% and 88%, respectively. A sensitivity of 86% and specificity of 86% was obtained with the commercial kit. Conclusion: Detection of anti-H. pylori specific IgG in oral fluid by ELISA is comparable in sensitivity and specificity with serum based methods. Oral fluid based ELISA could provide a reliable, non-invasive method for the diagnosis of H. pylori infection. Saliva testing may have a role in epidemiological studies. Keywords: Helicobacter pylori, ELISA, Oral fluid

  8. High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

    Science.gov (United States)

    Urusov, Alexandr E; Petrakova, Alina V; Gubaydullina, Milyausha K; Zherdev, Anatoly V; Eremin, Sergei A; Kong, Dezhao; Liu, Liqiang; Xu, Chuanlai; Dzantiev, Boris B

    2017-05-01

    To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration. The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

  9. Persistent Low Toxoplasma IgG Avidity Is Common in Pregnancy: Experience from Antenatal Testing in Norway.

    Directory of Open Access Journals (Sweden)

    Gry Findal

    Full Text Available The parasite Toxoplasma gondii might harm the fetus if a woman is infected during pregnancy. IgG seroconversion and significant increase in IgG antibody amount in pregnancy indicates maternal infection. Presence of toxoplasma immunoglobulin M (IgM, immunoglobulin G (IgG and low IgG avidity in a single serum sample indicates possible maternal infection, but positive toxoplasma IgM and low IgG avidity may persist for months and even years. We aimed to evaluate avidity development during pregnancy in a retrospective study. Serial blood samples from 176 pregnant women admitted to Oslo University Hospital 1993-2013 for amniocentesis because of suspected toxoplasma infection were included. Data were obtained from journals and laboratory records. The avidity method used was based on Platelia Toxo IgG assay. Mean maternal age at first serology was 29.9 years (SD 5.2, range 18-42. In 37 (21% women only the avidity increased from low to high in < 3 months. In 139 (79% the IgG avidity remained below the high threshold ≥ 3 months and within this group 74 (42% women had stable low IgG avidity during the observation period. Median gestational age at first test was 10.6 weeks (range 4.6-28.7. Fetal infection was detected in four children, but none among children whose mother had stable low IgG avidity. The first antenatal toxoplasma serology should ideally be collected in early pregnancy and if stable values of toxoplasma IgM and low IgG-avidity are detected in a second sample after three to four weeks, the need for amniocentesis can be questioned.

  10. Anticardiolipin antibodies in classic pediatric hemolytic-uremic syndrome: a possible pathogenic role.

    Science.gov (United States)

    Ardiles, L G; Olavarría, F; Elgueta, M; Moya, P; Mezzano, S

    1998-01-01

    Anticardiolipin (aCL) antibodies have been associated with thrombocytopenia, hemolytic anemia and an increased risk of thrombosis in different vascular locations, even in the absence of lupus. The classic hemolytic-uremic syndrome is a postinfectious acute renal failure characterized by hemolytic anemia, thrombocytopenia and the presence of widespread glomerular thrombosis in the kidney, with pathogenic mechanisms that remain to be identified. In order to establish the frequency of aCL antibodies in this syndrome and to identify a possible role in the pathogenesis and clinical manifestations, 17 patients were studied during the reactant phase of the disease looking for an association between the presence of aCL antibodies (isotypes IgG, IgA and IgM) and the main clinical variables of the syndrome. In 8 patients IgG aCL was present, 2 patients had IgM aCL, and 1 had IgA antibodies on the solid-phase ELISA aCL assays, but no association could be demonstrated with the clinical variables studied. Although it might correspond to an epiphenomenon related to the triggering intestinal infection, a pathogenic role cannot be discarded and additional studies should be performed.

  11. Antiphosphatidylserine/prothrombin antibodies (aPS/PT) as potential markers of antiphospholipid syndrome.

    Science.gov (United States)

    Vlagea, Alexandru; Gil, Antonio; Cuesta, Maria V; Arribas, Florencia; Diez, Jesús; Lavilla, Paz; Pascual-Salcedo, Dora

    2013-06-01

    The antiphospholipid antibodies present in antiphospholipid syndrome (APS) are directed at a number of phospholipid-binding proteins: β2 glycoprotein I (β2GPI), prothrombin, and so on. Antibodies directed at β2GPI are accepted as a classification criterion for APS, while the presence of antiprothrombin antibodies is not. In the present article, we investigated the possible role of antiphosphatidylserine/prothrombin antibodies (aPS/PT) as marker of APS on a cohort of 295 individuals with APS (95 primary APS and 45 secondary APS) and APS-related diseases. We found aPS/PT to be highly associated with venous thrombosis (immunoglobulin G [IgG] aPS/PT odds ratio [OR], 7.44; 95% confidence interval [CI], 3.97-13.92 and IgM aPS/PT OR, 2.54; 95% CI, 1.35-4.77) and obstetric abnormalities (IgG aPS/PT OR, 2.37; 95% CI, 1.04-5.43), but not with arterial thrombosis. A very high degree of concordance between the concentration of aPS/PT and lupus anticoagulant activity was demonstrated. Therefore, we support the inclusion of aPS/PT determination as second-level assay to confirm APS classification.

  12. Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

    International Nuclear Information System (INIS)

    Paunkovic, N.; Paunkovic, J.

    2003-01-01

    Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I -labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)

  13. Evaluation of the Chagas Stat-Paktm Assay for Detection of Trypanosoma cruzi Antibodies in Wildlife Reservoirs

    Science.gov (United States)

    Yabsley, Michael J.; Brown, Emily L.; Roellig, Dawn M.

    2010-01-01

    An immunochromatographic assay (Chagas Stat-Pak™) was evaluated for the detection of Trypanosoma cruzi antibodies in 4 species of wildlife reservoirs. Antibodies to T. cruzi were detected in raccoons (Procyon lotor) (naturally and experimentally infected) and degus (Octodon degu) (experimentally-infected) using the Chagas Stat-Pak. In naturally exposed wild raccoons, the Chagas Stat-Pak had a sensitivity and specificity of 66.7–80.0% and 96.3%, respectively. Compared with indirect immunofluorescent antibody assay results, serocon-version as determined by Chagas Stat-Pak was delayed for experimentally infected raccoons, but occurred sooner in experimentally infected degus. The Chagas Stat-Pak did not detect antibodies in naturally or experimentally infected Virginia opossums (Didelphis virginiana) or in experimentally infected short-tailed opossums (Monodelphis domestica). These data suggest that the Chagas Stat-Pak might be useful in field studies of raccoons and degus when samples would not be available for more-conventional serologic assays. Because this assay did not work on either species of marsupial, the applicability of the assay should be examined before it is used in other wild species. PMID:19016578

  14. Preparation of antibody coated tubes

    International Nuclear Information System (INIS)

    Robles Berrueta, A.M.

    1997-01-01

    Full text: 1. Purification of IgG: 2-4 ml serum at pH 8 with Buffer tris 1M pH 8. Let serum pass through the column of Sepharose Prot. A (1-2 ml). Wash with: a) Buffer tris 0.1M pH 8; b) Buffer tris 0.01M pH 8. Elute with Glycine 0.1M pH 3 adding eluant at 0.5 ml fractions and collect in eppendorf tubes containing 50μ1 Buffer tris 1M pH 8 to neutralize. 20 fractions are collected. Absorbency at 280nm is measured in each fraction. Pool is formed with protein factions. Dialysis against water is done during 48 hours changing water twice during that lapse. Regenerate column for future use with 1 wash Urea 2M, second with LiCl 1M and third wash with Glycine 0.1 M pH 2.5. 2. Antibody Immobilization on an Activated Solid Phase: NUNC maxisorp, Star tube 75x12 mm is trade mark for polystyrene tubes from Pharmacia with less than 5% CV% inhomogeneity in adsorption of IgG and less than 10% for random bias of any result from mean value. They are kept closed until use. They are not reusable. The antibody is diluted to a working dilution with buffer carbonate-bi carbonate 0.1M, pH 9.6 (BCBic). Adequate volume is pipetted into maxisorb NUNC tubes paying attention not to produce droplets (1/200 dilution and 0.3 ml/tube are used for TSH assays). An incubation overnight is enough to get maximum IgG binding. Antibody solution is recovered for further use (after mixing with additional antibody). Solid phase is subject to washing with phosphate buffer with non-Ionic detergent (1 ml PB.5 + 0.5% Tween 20) and then with pure water. Tubes are left two hours upside down and kept tightly closed with dissicant at - 20 deg. C

  15. Detection of antibodies in human serum using trimellityl-erythrocytes: direct and indirect haemagglutination and haemolysis.

    Science.gov (United States)

    Turner, E S; Pruzansky, J J; Patterson, R; Zeiss, C R; Roberts, M

    1980-02-01

    Utilizing trimellityl-erythrocytes (TM-E), antibodies were detected in sera of seven workers with trimellitic anhydride (TMA) induced airway syndromes by direct haemagglutination, indirect haemagglutination with anti-human IgG, IgA or IgM or by haemolysis. Detectable levels of antibody were obtained with all three methods. The most sensitive technique was indirect haemagglutination using anti-IgG. When added as an inhibitor, TM-human serum albumin produced a 10- to 800-fold reduction in titres. TM-ovalbumin of similar epitope density was less inhibitory and sodium trimellitate the least inhibitory on a molar basis. All of the assays using haptenized human red cells were also capable of detecting anti-TM antibodies in Rhesus monkeys whose airways had been exposed to TMA. These assays are useful for detecting anti-TM antibodies and may also be adapted to demonstrate antibodies induced against other inhaled haptens in sera of environmentally exposed individuals or in animal models of such exposure.

  16. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.

    2007-01-01

    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery. ...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

  17. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Science.gov (United States)

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

  18. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Directory of Open Access Journals (Sweden)

    Masaki Kurogochi

    Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

  19. Antibody responses of swine following infection with Mycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and M. flocculare.

    Science.gov (United States)

    Gomes Neto, João Carlos; Strait, Erin L; Raymond, Matthew; Ramirez, Alejandro; Minion, F Chris

    2014-11-07

    Several mycoplasma species possessing a range of virulence have been described in swine. The most commonly described are Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, and Mycoplasma flocculare. They are ubiquitious in many pig producing areas of the world, and except for M. hyopneumoniae, commercial antibody-based assays are lacking for most of these. Antibody cross-reactivity among these four mycoplasma species is not well characterized. Recently, the use of pen-based oral fluids for herd surveillance is of increasing interest. Thus, this study sought to measure pig antibody responses and the level of cross-reactivity in serum and pen-based oral fluids after challenge with four species of swine mycoplasmas. Four groups of four mycoplasma-free growing pigs were separately inoculated with the different mycoplasma species. Pen-based oral fluids and serum samples were collected weekly until necropsy. Species-specific Tween 20 ELISAs were used to measure antibody responses along with four other commercial M. hyopneumoniae ELISAs. Animals from all groups seroconverted to the challenge species of mycoplasma and no evidence of cross-contamination was observed. A delayed antibody response was seen with all but M. hyorhinis-infected pigs. Cross-reactive IgG responses were detected in M. hyopneumoniae- and M. flocculare-infected animals by the M. hyorhinis Tween 20 ELISA, while sera from M. hyosynoviae and M. flocculare-infected pigs were positive in one commercial assay. In pen-based oral fluids, specific anti-M. hyopneumoniae IgA responses were detected earlier after infection than serum IgG responses. In summary, while some antibody-based assays may have the potential for false positives, evidence of this was observed in the current study. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  1. Egg Yolk Antibodies, State of the Art and Future Prospects.

    Science.gov (United States)

    Schade, Rüdiger; Hlinak, Andreas

    1996-01-01

    Immunization of chickens and extraction of antibodies from egg yolk belongs to the alternative methods since the animals suffering is reduced by non-invasive antibody-sampling. Also, the number of animals needed to produce a certain amount of antibody can be reduced since chickens produce a significant higher antibody quantity than rabbits. Despite its several advantages this technology (IgY-technology) is rather scarcely used. Traditional behavior as well as limited or no information at all may hamper a broader acceptance at present. However, significant arguments exist in chicken housing, the choice of appropriate IgY-extraction methods and a lack of information regarding the use of IgY-antibodies. This paper intends to give a short introduction in the IgY-technology, to briefly discuss the state of the art and to inform on recent developments and discussions in this field. The suitability of IgY for special fields of application (as a result of the structural differences between IgY and IgG) is emphasized (e.g. assays combining IgG and IgY, immunization of chickens against highly conserved anti-genes). In addition, it is stressed that the IgY-technology as an alternative method can particularly integrate requirements of animal protection (reduce, replace, refine), science (characteristics of avian immune system and resulting properties of IgY) and economy (amount of IgY produced from one chicken).

  2. Autoreactive lymphocytes in thyroid disorders. 2. Comparison of anti-thyroglobulin antibody production by plaque-forming cell, radio-immunological and enzyme-linked immunosorbent assays

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, J; Feldt-Rasmussen, U; Siersbaek-Nielsen, K; Hoeier-Madsen, M; Larsen, F; Husby, S

    1986-01-01

    Blood mononuclear cells (MNC) from 9 randomly selected patients with autoimmune thyroiditis were stimulated in vitro with pokeweed mitogen (PWM), a polyclonal B lymphocyte activator. The secretion of immunoglobulins (Ig) and anti-thyroglobulin antibodies (TgAb) was assayed by means of haemolytic plaque-forming cell (PFC) assays, radioimmune assay (RIA) and enzyme-linked immunosorbent assays (ELISA). Total Ig and TgAb production was maximal using MNC cultured at 1.0 x 10/sup 6//ml as estimated by PFC, RIA and ELISA. The Ig and TgAb production as measured by RIA and ELISA was 1.5 - 3 times higher after 12 days' culture compared to 6 days' culture. Ig and TgAb production measured by PFC-assays at day 6 correlated positively to the results obtained by RIA and ELISA at day 12. PWM-induced TgAb secretion correlated positively to TgAb titres in serum. As judged by PFC, TgAb production was found in 8/9 patients; about 5% (range 0 - 7.9%) of the total PWM-stimulated IgG-secreting cells were involved in TgAb secretion. TgAb production as measured by ELISA and RIA was found in 6/9 patients. By reference to an affinity-purified human TgAb preparation, the TgAb secretion was about 0.7% (range 0 - 21.3%) of the total PWM-induced IgG secretion.

  3. Quantitative cumulative biodistribution of antibodies in mice

    Science.gov (United States)

    Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

    2014-01-01

    The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level. PMID:24572100

  4. Kinetics of IgG antibody to cytomegalovirus (CMV after birth and seroprevalence of anti-CMV IgG in Chinese children

    Directory of Open Access Journals (Sweden)

    Chen Jie

    2012-12-01

    Full Text Available Abstract Background Prevalence of cytomegalovirus (CMV infection is 90–100% in developing countries; however, the kinetics of anti-CMV IgG in infants remains elusive. Methods Sera from 112 mother-newborn pairs and longitudinal samples from 41 infants up to 2-year old were tested for anti-CMV IgG and IgM. Additionally, samples from 837 healthy children were included. Results Of 112 mothers, 108 (96.4% were anti-CMV IgG positive; their 108 newborns were also seropositive. In a 2-year follow-up among 40 infants of positive mothers, anti-CMV IgG level in 8 individuals decreased with time and became undetectable by age of 3.5–8 months, and that in 32 others decreased at 1- and 3.5-month old, and then i